Cannabidinoid derivatives

ABSTRACT

The disclosure relates to cannabinoid derivative compounds, pharmaceutical compositions made thereof, and methods for treating various diseases and disorders including cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/565,438, filed on Nov. 30, 2011, the disclosure of which isincorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was funded by Grant Nos. CA 102412 and CA111723 awardedby the National Institutes of Health. The government has certain rightsin the invention.

TECHNICAL FIELD

This invention relates to cannabidiol and derivatives thereof,pharmaceutical compositions made therefrom, and methods of treatment forvarious disorders, including neoplastic disorders.

BACKGROUND

The hemp plant Cannabis sativa, commonly referred to as marijuana, hasbeen used to ameliorate symptoms of diseases and disorders for thousandsof years. Currently an oral formulation of Δ⁹-tetrahydrocannabinol, theprimary active cannabinoid constituent of marijuana, is approved as ananti-emetic agent for treating cancer patients undergoing chemotherapy.Additional studies suggest that cannabinoids may increase appetite andalleviate pain in the same patient population.

SUMMARY

In a particular embodiment, the disclosure provides for a compoundhaving the structure of Formula II:

or a pharmaceutically acceptable salt, or prodrug thereof, wherein:

X is independently a C or N;

R²-R³ are each independently selected from the group consisting ofhydroxyl, (C₁-C₂)alkoxy, carboxylic acid, amine, halo, cyano, and(C₁-C₃) ester;

R⁴-R⁵ are each independently selected from the group consisting ofhydrogen, deuterium, hydroxyl, (C₁-C₂)alkoxy, carboxylic acid, amine,halo, cyano, and (C₁-C₃)ester;

R⁶ is selected from the group consisting of an unsubstituted (C₁-C₁₂)alkyl, an unsubstituted hetero (C₁-C₁₁) alkyl, an unsubstituted (C₁-C₁₂)alkenyl, an unsubstituted hetero (C₁-C₁₁) alkenyl, an unsubstituted(C₁-C₁₂)alkynyl, and an unsubstituted hetero(C₁-C₁₁)alkynyl;

R¹⁰-R¹⁹ are each independently selected from the group consisting ofhydrogen, deuterium, functional group (“FG”), optionally substituted(C₁-C₈)alkyl, optionally substituted hetero(C₁-C₈)alkyl, optionallysubstituted (C₁-C₈)alkenyl, optionally substituted hetero(C₁-C₈)alkenyl,optionally substituted (C₁-C₈)alkynyl, optionally substitutedhetero(C₁-C₈)alkynyl, optionally substituted (C₅-C₁₂)cycloalkyl,optionally substituted (C₅-C₁₂)cycloalkenyl, optionally substituted(C₅-C₁₂)cycloalkynyl, optionally substituted (C₄-C₁₁)heterocycle,optionally substituted aryl, and optionally substituted extended mixedring system; and

R²⁰ is selected from the group consisting of optionally substituted(C₅-C₁₂)cycloalkyl, optionally substituted (C₅-C₁₂)cycloalkenyl,optionally substituted (C₅-C₁₂)cycloalkynyl, optionally substituted(C₄-C₁₁)heterocycle, substituted aryl, optionally substituted arylhaving two or more rings, and optionally substituted extended mixed ringsystem.

In a further embodiment, R⁶ is selected from the group consisting ofmethyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl,isopropyl, sec-butyl, (1-methyl)butyl, (1-methyl)pentyl,(1-methyl)hexyl, (1-methyl)heptyl, (1,1-dimethyl)propyl,(1,1-dimethyl)butyl, (1,1-dimethyl)pentyl, (1,1-dimethyl)hexyl,(1,1-dimethyl)heptyl, (1,2-dimethyl)propyl, (1,2-dimethyl)butyl,(1,2-dimethyl)pentyl, (1,2-dimethyl)hexyl, (1,2-dimethyl)heptyl,(1,3-dimethyl)butyl, (1,3-dimethyl)pentyl, (1,3-dimethyl)hexyl,(1,3-dimethyl)heptyl, (1,4-dimethyl)pentyl, (1,4-dimethyl)hexyl,(1,4-dimethyl)heptyl, (1,5-dimethyl)hexyl, (1,5-dimethyl)heptyl,(1,6-dimethyl)heptyl, (1,2-diethyl)butyl, (1,2-diethyl)pentyl,(1,2-diethyl)hexyl, (1,2-diethyl)heptyl, (1,2-diethyl)pentyl,(1,3-diethyl)pentyl, (1,3-diethyl)hexyl, (1,3-diethyl)heptyl,(1,4-diethyl)pentyl, (1,4-diethyl)hexyl, (1,4-diethyl)heptyl,(1,5-diethyl)hexyl, (1,5-diethyl)heptyl, (1,6-diethyl)heptyl,(1,2,3-trimethyl)butyl, (1,1,2-trimethyl)butyl, (1,1,3-trimethyl)butyl,(1,2,3-trimethyl)pentyl, (1,1,2-trimethyl)pentyl,(1,1,3-trimethyl)pentyl, (1,2,4-trimethyl)pentyl,(1,3,4-trimethyl)pentyl, (1,1,4-trimethyl)pentyl,(1,2,3-trimethyl)hexyl, (1,1,2-trimethyl)hexyl, (1,1,3-trimethyl)hexyl,(1,2,4-trimethyl)hexyl, (1,2,5-trimethyl)hexyl, (1,1,4-trimethyl)hexyl,(2,3,4-trimethyl)hexyl, (2,3,5-trimethyl)hexyl, (1,1,5-trimethyl)hexyl,(1,2,3-trimethyl)heptyl, (1,1,2-trimethyl)heptyl,(1,1,3-trimethyl)heptyl, (1,2,4-trimethyl)heptyl,(1,1,5-trimethyl)heptyl, (1,1,6-trimethyl)heptyl,(1,2,5-trimethyl)heptyl, (1,2,6-trimethyl)heptyl,(2,3,4-trimethyl)heptyl, (2,3,5-trimethyl)heptyl,(2,3,6-trimethyl)heptyl, (2,4,5-trimethyl)heptyl,(2,4,6-trimethyl)heptyl, (3,4,5-trimethyl)heptyl,(3,4,6-trimethyl)heptyl, and (4,5,6-trimethyl)heptyl.

In yet a further embodiment, R²⁰ is selected from the group consistingof an optionally substituted (C₅-C₇)cycloalkyl, an optionallysubstituted (C₅-C₇)cycloalkenyl, a substituted aryl, an optionallysubstituted aryl having two or more rings, and an optionally substitutedheterocycle containing 4, 5, or 6 ring atoms.

Examples of optionally substituted heterocycles containing 4, 5, or 6ring atoms include:

Examples of substituted aryls and optionally substituted aryls havingtwo or more rings include:

Examples of optionally substituted (C₅-C₇)cycloalkyls include:

In a certain embodiment, a compound of Formula II, may further have astructure of Formula III:

or a pharmaceutically acceptable salt, or prodrug thereof, wherein:

X is independently either a C or N;

R²-R³ are each independently a hydroxyl or (C₁-C₂)alkoxy;

R⁶ is selected from the group consisting of an unsubstituted (C₁-C₁₂)alkyl, an unsubstituted hetero (C₁-C₁₁) alkyl, an unsubstituted (C₁-C₁₂)alkenyl, an unsubstituted hetero (C₁-C₁₁) alkenyl, an unsubstituted(C₁-C₁₂)alkynyl, and an unsubstituted hetero(C₁-C₁₁)alkynyl;

R¹¹-R¹⁸ are each independently selected from the group consisting ofhydrogen, deuterium, FG, optionally substituted (C₁-C₈)alkyl, optionallysubstituted hetero(C₁-C₈)alkyl, optionally substituted (C₁-C₈)alkenyl,optionally substituted hetero(C₁-C₈)alkenyl, optionally substituted(C₁-C₈)alkynyl, optionally substituted hetero (C₁-C₈) alkynyl; and

R²¹-R³¹ are each independently selected from the group consisting ofhydrogen, deuterium, FG, optionally substituted (C₁-C₈)alkyl, hetero(C₁-C₈) alkyl, optionally substituted (C₁-C₈)alkenyl, optionallysubstituted hetero(C₁-C₈)alkenyl, optionally substituted (C₁-C₈)alkynyl, optionally substituted hetero (C₁-C₈) alkynyl, optionallysubstituted (C₅-C₈)cycloalkyl, optionally substituted(C₅-C₈)cycloalkenyl, optionally substituted (C₅-C₈)cycloalkynyl,optionally substituted (C₄-C₈)heterocycle, optionally substituted aryl,and optionally substituted extended mixed ring system.

In a further embodiment, a compound of Formula II or of Formula III hasthe structure of:

or a pharmaceutically acceptable salt, or prodrug thereof. Examples ofprodrugs for a compound of:include:

wherein, X is a pharmaceutically acceptable counter ion.

In another embodiment, a compound of Formula II or of Formula III hasthe structure selected from the group of:

In a particular embodiment, the disclosure provides for pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier togetherwith a compound of the disclosure. In a further embodiment, thepharmaceutical composition may further comprise an additionaltherapeutic agent, such as Δ⁹-tetrahydrocannabinol (“THC”) or a THCderivative. Examples of derivatives of THC includeΔ⁹-tetrahydrocannabinol-C₄, Δ⁹-tetrahydrocannabivarin,tetrahydrocannabiorcol, Δ⁹-tetrahydro-cannabinolic acid A,Δ⁹-tetrahydro-cannabinolic acid B, Δ⁹-tetrahydro-cannabinolic acid-C₄ A,Δ⁹-tetrahydro-cannabinolic acid-C₄, Δ⁹-tetrahydro-cannabivarinic acid A,Δ⁹-tetrahydro-cannabiorcolic acid A, Δ⁹-tetrahydro-cannabiorcolic acidB, (−)-Δ⁸-trans-(6aR,10aR)-Δ⁸-tetrahydrocannabinol,(−)-Δ⁸-trans-(6aR,10aR)-tetrahydrocannabinolic acid A, and(−)-(6aS,10aR)-Δ⁹-tetrahydrocannabinol. In yet a further embodiment, anadditional therapeutic agent is selected from alkylating agents, cancerimmunotherapy monoclonal antibodies, anti-metabolites, mitoticinhibitors, anti-tumor antibiotics, topisomerase inhibitors,photosensitizers, tyrosine kinase inhibitors, anti-cancer agents,chemotherapeutic agents, anti-migraine treatments, anti-tussives,mucolytics, decongestants, anti-allergic non-steroidals, expectorants,anti-histamine treatments, anti-retroviral agents, CYP3A inhibitors,CYP3A inducers, protease inhibitors, adrenergic agonists,anti-cholinergics, mast cell stabilizers, xanthines, leukotrieneantagonists, glucocorticoid treatments, antibacterial agents, antifungalagents, sepsis treatments, steroidals, local or general anesthetics,NSAIDS, NRIs, DARIs, SNRIs, sedatives, NDRIs, SNDRIs, monoamine oxidaseinhibitors, hypothalamic phoshpholipids, anti-emetics, ECE inhibitors,opioids, thromboxane receptor antagonists, potassium channel openers,thrombin inhibitors, growth factor inhibitors, anti-platelet agents,P2Y(AC) antagonists, anti-coagulants, low molecular weight heparins,Factor VIa inhibitors, Factor Xa inhibitors, renin inhibitors, NEPinhibitors, vasopepsidase inhibitors, squalene synthetase inhibitors,anti-atherosclerotic agents, MTP inhibitors, calcium channel blockers,potassium channel activators, alpha-muscarinic agents, beta-muscarinicagents, anti-arrhythmic agents, diuretics, thrombolytic agents,anti-diabetic agents, mineralocorticoid receptor antagonists, growthhormone secretagogues, aP2 inhibitors, phophodiesterase inhibitors,anti-inflammatories, anti-proliferatives, antibiotics, farnesyl-proteintransferase inhibitors, hormonal agents, plant-derived products,epipodophyllotoxins, taxanes, prenyl-protein transferase inhibitors,anti-TNF antibodies and soluble TNF receptors, and Cyclooxygenase-2inhibitors. In another embodiment, the additional therapeutic agent isselected from alkylating agents, cancer immunotherapy monoclonalantibodies, anti-metabolites, mitotic inhibitors, anti-tumorantibiotics, topisomerase inhibitors, photosensitizers, tyrosine kinaseinhibitors, anti-cancer agents, and chemotherapeutic agents. In yetanother embodiment, the additional therapeutic agent is an anti-canceragent, such as paclitaxel and/or temozolomide.

In a particular embodiment, the disclosure provides a method forinhibiting Id-1 expression, cell proliferation, cell invasion,metastasis or a combination thereof in vivo and/or in vitro byadministering a compound disclosed herein.

In a certain embodiment, the disclosure provides a method for treating adisease or disorder in a subject, comprising administering to a subjecta therapeutically effective amount of a compound disclosed herein,wherein the disease or disorder can be ameliorated by inhibiting theexpression of an Id polypeptide, by agonizing cannabinoid type 2 (“CB₂”)receptors or a combination thereof. In a further embodiment, the diseaseor disorder is selected from the group of cancer, chronic pancreatitis,psoriasis, neoplasms, angiomas, endometriosis, obesity, age-relatedmacular degeneration, retinopathies, restenosis, scaring, fibrogenesis,fibrosis, cardiac remodeling, pulmonary fibrosis, scleroderma, failureassociated with myocardial infarction, keloids, fibroid tumors,stenting, Alzheimer's Disease, Parkinson's Disease, age relateddementia, Huntington's Disease, and amyotrophic lateral sclerosis.Examples of cancer which can be treated with a compound disclosed hereininclude leukemia, melanoma, squamous cell carcinoma (SCC),hepatocellular carcinoma, colorectal adenocarcinoma, pancreatic cancer,lung cancer, kidney cancer, medullary thyroid cancer, papillary thyroidcancer, astrocytic tumor, neuroblastoma, Ewing's sarcoma, ovarian tumor,cervical cancer, endometrial carcinoma, breast cancer, prostate cancer,and malignant seminoma. In a certain embodiment, the cancer to betreated is a breast cancer or a brain cancer, such as glioblastomamultiforme. In another embodiment, the disclosure provides methods oftreating chronic pain, neuropathic pain and neuropathies, spinal cordand head injuries, and radiation injury (e.g., following cancertreatment).

In a particular embodiment, a method of treating a disease or disorderin a subject comprises administering to a subject a therapeuticallyeffective amount of a compound having the structure of:

or a pharmaceutically acceptable salt, or prodrug thereof, wherein thedisease or disorder can be ameliorated by inhibiting the expression ofan Id polypeptide, by activating CB₂ receptors or a combination thereof.In a further embodiment, the disease or disorder is selected from thegroup of leukemia, melanoma, SCC, hepatocellular carcinoma, colorectaladenocarcinoma, pancreatic cancer, lung cancer, kidney cancer, medullarythyroid cancer, papillary thyroid cancer, astrocytic tumor,neuroblastoma, Ewing's sarcoma, ovarian tumor, cervical cancer,endometrial carcinoma, breast cancer, prostate cancer, and malignantseminoma. In yet a further embodiment, the disease or disorder is abreast cancer or a brain cancer, such as glioblastoma multiforme. Inanother embodiment, the method of treating a disease or disorder with acompound disclosed herein further comprises administering to the subjectone or more alkylating agents, cancer immunotherapy monoclonalantibodies, anti-metabolites, mitotic inhibitors, anti-tumorantibiotics, topoisomerase inhibitors, photosensitizers, tyrosine kinaseinhibitors, anti-cancer agents, chemotherapeutic agents, or acombination thereof.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the description anddrawings, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of this specification, illustrate one or more embodiments of thedisclosure and, together with the detailed description, serve to explainthe principles and implementations of the disclosure.

FIG. 1A-E provides data indicating that cannabidiol (“CBD”) is aneffective inhibitor of Id-1 expression, and thereby inhibitsproliferation and invasiveness of MDA-MD231 breast cancer cells. (A)Depicts the results of a Boyden chamber invasion assay used to determinethe effects of cannabinoids on the invasiveness of aggressive humanbreast cancer MDA-MB231 cells. Compounds were added at concentrations of0.1 μM, 1.0 μM, or 1.5 μM. Data are presented as relative invasivenessof the cells through the Matrigel, where the respective controls are setas 100%. (B) Depicts Western blot analysis of proteins from MDA-MB231cells treated with vehicle (control), 0.1 μM, 1.0 μM, or 1.5 μM of CBDfor three days and analyzed as described below. (C) Provides a graphdepicting the relative expression of Id-1 in treated cells/vehiclecells. Proteins from MDA-MB231 cells treated with additionalcannabinoids for three days were extracted and analyzed for Id-1expression by Western blot analysis. Normalization was carried out bystripping the blots and re-probing with a monoclonal anti-tubulinantibody. (D) Presents a graph depicting the inhibitory effect of 1.5 μMCBD on Id-1 expression compared over a time course of one-, two-, andthree-days. (E) Presents data depicting the structure activityrelationship of cannabinoids and the regulation of Id-1 proteinexpression. Proteins from MDA-MB231 cells treated with vehicle (control)or 1.5 μM of various cannabinoid compounds for two days were thenanalyzed for Id-1 expression by Western blot analysis. A high molecularweight non-specific band was used as a loading control (LC). Data arethe mean of at least three replicates, bars±SE. Data were compared usinga one-way ANOVA with a corresponding Dunnett's post-hoc test. (*)indicates statistically significant differences from control (p<0.05).

FIG. 2A-C presents data indicating that ectopic expression of Id-1blocks the effect of CBD on MDA-MB231 invasiveness. (A)

Provides representative light microscope images of control MDA-MB231cells ((−)Id-1 cells, upper panels) and of MDA-MB231 cells thatectopically express Id-1 ((+)Id-1 cells, lower panels) that were treatedwith vehicle (control) or CBD (1.5 μM CBD) for two days, followed by aninvasion assay performed overnight. (B) Provides data showing therelative invasiveness of the cells through the Matrigel, where therespective controls are set as 100%, and are the mean of at least threereplicates, bars±SE. Data were compared using the unpaired Student'st-test. (*) indicates statistically significant differences from control(p<0.05). (C) Presents a Western blot showing the inhibitory effect ofCBD on Id-1 expression in (−)Id-1 MDA-MB231 cells in comparison to(+)Id-1 MDA-MB231 cells.

FIG. 3A-C provides data indicating that CBD inhibits the expression ofId-1 gene at the mRNA and promoter levels in MDA-MB231 cells. (A)Presents the inhibition of the Id-1 gene product (434 bp) by CBD.Expression of the β-actin gene product (232 bp) was used as a control.(B) Provides luciferase activity in MDA-MB231 cells transientlytransfected with Id-1-sbsluc as determined in the presence of vehicle(control) or CBD (1.5 μM). Cells were treated for 2 days and luciferaseactivity was measured. (C) Presents data for cells treated for 3 days.For both (B) and (C), all values were normalized for the amount of β-galactivity present in the cell extracts. Data are the mean of at leastthree replicates, bars±SE. The data are represented as percentage ofactivity of the treated cells/vehicle cells×100. Data were comparedusing the unpaired Student's t-test. (*) indicates statisticallysignificant differences from control (p<0.05).

FIG. 4A-C provides that CBD produced a dose-dependent reduction ofmetastatic spread to the lung and increased the survival rate of mice.Lung metastases were generated in BALB/c mice by tail vein injection of2×10⁴ 4T1 cells. (A) One day after the injection, the tumor bearing micewere injected i.p. once a day with vehicle or CBD (0.1 to 5 mg/kg) for14 days and percent metastasis was evaluated. Percent metastasis=totaltumor number of lung metastatic foci in drug treated group/total numberof lung metastatic foci in vehicle treated group where the respectivecontrols (vehicle treated cells) were set as 100%. (B) Lung metastasesmeasured in mice treated with vehicle or CBD 1 mg kg⁻¹ included those <2mm and 2 mm. (C) Mice treated with vehicle or 1 mg kg⁻¹ CBD, starting aday after tail vein injection of 2×10⁴ 4T1 cells, were observed untilthey demonstrated signs of disease progression that necessitatedeuthanasia. Survival between groups was compared using and Kaplan-Meieranalysis. **p<0.01 (unpaired Student't-test) and p<0.0001 (one-wayANOVA; *p<0.05, ^(#)p<0.001 Dunnett's post-hoc test).

FIG. 5A-C demonstrates that CBD decreases Id-1 expression and Ki-67staining in lung metastatic foci. (A) Immunohistochemical detection ofId-1 and Ki67 was performed in lung tissues of vehicle (left) and CBD(right) treated 4T1-derived tumors. Nuclei are visible in blue(hematoxylin staining). Pictures are x400 magnification. (B) Theintensity of Id-1 expression is shown as follows: □, Negative;

, Weakly positive; ▪, Strongly positive. The data are presented as astatistical analysis (lower panel). (C) The percentage of Ki67 positivecells per lung metastatic foci was also evaluated. *p<0.002 (unpairedStudent's t-test).

FIG. 6A-C provides that CBD inhibited Id-1 and Ki67 expression in lungmetastatic foci. (A) Immunohistochemical detection of Id-1 and Ki67 wasperformed in lung tissues of Vehicle (left) and CBD (right) treatedmice. Nuclei are visible in blue (hematoxylin staining). Pictures ofupper panels are ×200 magnification, and lower panels are ×400. (B) Theintensity of the immunohistochemical (IHC) detection of Id-1 was gradedfrom 0 to 4, and the percentage of cells per foci for each intensitylevel is shown as follows: □, grade 0;

, grade 1;

, grade 2;

, grade 3; ▪, grade 4. (Right), the data are presented as a statisticalanalysis. (C) The percentage of Ki67 positive cells per lung metastaticfoci was evaluated.

FIG. 7A-D provides that CBD reduced the formation of metastatic foci andincreased the survival rate of mice in advanced stages of metastaticprogression. Lung metastases were generated in BALB/c mice after tailvein injection of 2×10⁴ 4T1 cells. (A) The pictures are representativeof tumor formation observed at day five, seven, and nine. (B) Seven daysafter the injection of tumor cells, mice were injected i.p. once a daywith vehicle, 0.5, 1 or 10 mg/kg CBD for seven days. % metastasis and(C) the number of lung metastatic foci±2 mm were compared betweenvehicle and CBD treated groups. p<0.05 (one-way ANOVA; *p<0.05, **p<0.01Dunnett's post-hoc test). (D) Mice treated with vehicle or 1 mg/kg CBD,starting seven days after tail vein injection of 2×10⁴ 4T1 cells, wereobserved until they demonstrated signs of disease progression thatnecessitated euthanasia. Survival between groups was compared usingKaplan-Meier analysis.

FIG. 8A-C presents data showing CDB produces a dose-dependent inhibitionof tumor proliferation in-vivo. Tumors were generated by (A) injectionof 5×10⁵ 4T1 cells in the mammary fat pad of BALBc mice, or (B) bysubcutaneous injection of 2×10⁶ U251 cells into the flank of Nude mice.Daily treatment (systemic in (A) and peritumoral in (B) with CBD (mg/kg)was initiated one week after the initial injection of the cancer cells.The primary tumor volume was calculated by measuring the perpendicularlargest diameters of the tumor with a caliper and using the formula(L×W²/2). Representative samples show (C) that CBD eradicated a tumor inone of the NUDE mice.

FIG. 9 provides data showing the anti-metastatic activity of CBD againsthuman breast cancer in a xenograph mouse model. Lung metastases weregenerated in Nude mice after tail vein injection of 1×10⁶MDA-MB231-luc-D3H2LN cells. One day after the injection, the tumorbearing mice were injected i.p. once a day with vehicle or 0.5 mg/kg and1 mg/kg CBD for three weeks. Visible lung metastases were counted andmeasured by using a dissecting microscope. Data are represented aspercent metastasis compared to control (vehicle) where the control has100% metastasis.

FIG. 10A-B presents that CBD inhibited the formation of metastatic fociin later stages of metastatic progression. (A) Lung metastases weregenerated in BALB/c mice after tail vein injection of 20×10⁵ mouse 4T1cells. 1 mm³ tumors could be visuallized through a dissecting scope onday 7, therefore, this was chosen as the time point to innitiatetreatments. (B) One week after the tail vein injection of 20×10⁵ mouse4T1 cells, the tumor bearing mice were injected i.p. once a day withvehicle or 1 mg/kg and 10 mg/kg CBD for seven days. Visible lungmetastases were counted and measured by using a dissecting microscope.Data are represented as percent metastasis compared to control (vehicle)where the control has 100% metastasis.

FIG. 11A-C presents data showing that at optimal combined ratios, CBDcan enhance the ability of Paclitaxel to inhibit the viability of 4T1breast cancer cells. (A) Concentration response curves were generatedfor Paclitaxel (PAC) and CBD alone and in combination. (B) Theinhibitory values from the concentration response curves were used tocalculate combination index (CI) values at multiple combination ratios.(C) These data were also used to calculate IC₅₀ values, the slope of thecurve (m) and a goodness of fit value (r). Methods: Multiple viabilityassays in a 96 well format were run for each compound and the averagepercent inhibition of cell viability was calculated and transformed tofraction affected (Fa) e.g., percent inhibitory effect. Additional CIvalues and a dose reduction index (DRI) were also calculated usingCompusyn software W. After determining the (IC₅₀/Fa_(0.5)) values of thedrugs individually (C), components were then combined (B) at thefollowing concentration ranges: controls, 0.125×IC₅₀, 0.25×IC₅₀,0.5×IC₅₀ and a combination index was calculated where CI<1, =1, and >1indicate synergism, additive effect, and antagonism, respectively.

FIG. 12 provides a blot showing that treatment of U251 cells with CBDled to a concentration-dependent inhibition of Id-1 protein expression.

FIG. 13A-D provides data demonstrating that Id-1 protein expression isdown-regulated by CBD in breast, prostate, salivary gland, head and neckcancer cells as well as in glioblastoma cells. (A) Effect of CBD on Id-1protein expression in breast cancer cell lines, MDA-MB231 and MDA-MB436.(B) Effect of CBD on Id-1 protein expression in prostate cancer celllines, PC3 and DU145. (C) Effect of CBD on Id-1 protein expression insalivary gland and head and neck cancer cell lines, ACCM and SAS,respectively. (D) Effect of CBD on Id-1 protein expression ingliobastoma cancer cell lines, U251 and SF126. LC: loading control; Veh:cells treated with vehicle control; 1.5: cells treated with 1.5 μM CBD;and 2.5: cells treated with 2.5 μM CBD.

FIG. 14A-D provides that CBD inhibited glioblastoma multiforme (“GBM”)cell invasion. (A) 1×10⁵ GFP-labeled U251 cells were placed on top of a0.5 mm coronal slice of neonatal mouse brain and treated with vehicle(control) or CBD (1.0 μM) for 72 hours. Cells that migrated through theslice were counted using an inverted fluorescence microscope. Data areshown as mean number of cells in triplicate wells. Bars±SE, *p<0.05Student's t-test. The experiment was repeated three times with similarresults. Inset: representative samples of invading cells visualized fromthe bottom of the slice. (B) Tumors were generated in a xenograft mousemodel by intracranial injection of 0.5×10⁶ U251 cells. Each groupincluded five mice. Daily treatments with 15 mg/kg CBD were initiatedseven days after injection of the cells, and brains were harvested andsliced at the end of the treatments. (C) Panoramic viewer software(3DHISTECH) was used to measure the area of the tumor in the brain. (*)indicates statistically significant differences from control (p<0.02).(D) Representative sections demonstrate reduction in Id-1 (upper panel)and Ki67 (middle panel) expression in tumors responsive to CBDtreatment. Bar=100 μM. The insets (top panels) represent a 20 foldmagnification. Negative IgG controls (NC) are also shown.

FIG. 15A-D demonstrates the effect of CBD on the aggressiveness of headand neck as well as salivary gland cancer cells. (A) Head and neckcancer cell lines (SAS and HSC-2 in the upper panels) and salivary glandcancer cell lines (ACCM and ACC2 in the lower panels) were treated withCBD. Down-regulation of Id-1 gene expression was observed in all celllines. (B) Effect of CBD on SAS tumor cell viability and invasiveness isshown in the upper panels. Effect of CBD on ACCM tumor cell viabilityand invasiveness is shown in the lower panels. (C) Representativepictures of lung metastasis after injection of ACCM cells and dailytreatment with CBD. (D) The number of lung metastatic foci afterinjection of ACCM cells was significantly reduced upon CBD treatment.The total number of foci is shown in the left panel and the number ofmetastatic foci >1 mm is shown in the right panel.

FIG. 16A-C provides data showing that at optimal combined ratios, CBDcan enhance the ability of temozolomide to inhibit the viability of U251GBM cells. (A) Concentration response curves were generated fortemozolomide (“TMZ”) and CBD alone and in combination. (B) Theinhibitory values from the concentration response curves were used tocalculate combination index (CI) values at multiple combination ratioswhere CI<1, =1, and >1 indicate synergism, additive effect, andantagonism, respectively. (C) These data were used to calculate (i) IC₅₀values, the slope of the curve (m) and a goodness of fit value (r).Additionally, (ii) CI values and (iii) a dose reduction index (“DRI”)were calculated using Compusyn software.

FIG. 17 demonstrates that THC, CBD, and a combination of CBD and THCwere able to significantly reduce the invasiveness of U251 cells.

FIG. 18A-F provides data indicating that combinations of Δ⁹-THC and CBDproduced synergistic effects on the inhibition of cell growth in SF216and U251 cells but not in U87 cells. A 2×2 factorial design was used.(A) Depicts results for SF126. (B) Depicts results for U251. (C) Depictsresults for U87MG cells that were treated for three days with vehicle/nodrug, Δ⁹-THC, CBD, or a combination of Δ⁹-THC and CBD. Concentrations ofΔ⁹-THC and CBD that produce only minimal effects on cell proliferation(denoted low as opposed to high) were also tested in 2×2 factorialdesign in: (D) for SF126, and (E) for U251 cells. Cell proliferation wasmeasured using the MTT assay. The absorbance of the media alone at 570nm was subtracted, and percent control was calculated as the absorbanceof the treated cells/control cells×100. (F) Provides representativelight microscope images of the effects of the combination treatment onU251 cells from the experiment shown in (E), presented as (10×). Dataare the mean of at least 3 independent experiments, bars±SE.

FIG. 19A-C demonstrates that a combination treatment of Δ⁹-THC and CBDspecifically inhibited ERK activity. The effects of cannabinoids on MAPKactivity were analyzed using Western analysis. U251 cells were treatedwith vehicle or a combination of Δ9-THC (1.7 μM) and CBD (0.4 μM) fortwo days. Proteins were extracted and analyzed for (A) pERK and totalERK, and (B) pJNK 1/2 and p38 MAPK. U251 cells were treated with A9-THC(1.7 μM) and CBD (0.4 μM) alone and analyzed for (C) pERK and total ERK.Either α-tubulin or β-actin was used as a loading control (LC). Blotsare representative of at least 3 independent experiments.

FIG. 20 provides that the combination treatment of Δ⁹-THC and CBDproduced G1/S cell cycle arrest. Cell cycle was measured using PIstaining and FACS analysis. U251 cells were treated for three days withTHC (1.7 μM), CBD (0.4 μM), or a combination of THC (1.7 μM) and CBD(0.4 μM). Cells were collected and analyzed using a desktop FACS Caliburwith Cell Quest Pro software. Modfit was used to determine thepercentage of cell in G₀/G₁, S and G₂/G_(M) phase.

FIG. 21 presents data indicating that when combined, Δ⁹-THC and CBDproduced greater than additive effects on the cell cycle inhibition andinduction of apoptosis in U251 cells. U251 cells were treated for threedays with THC (1.7 μM), CBD (0.4 μM), or a 4:1 combination ratio [Δ⁹-THC(1.7 μM)/CBD (0.4 μM)]. The number of cells staining positive forannexin (apoptosis) were measured using FACS analysis. Percent controlwas calculated as positive annexin staining of the treated cells minuscontrol cells. Data are the mean of at least 3 independent experiments,bars±SE. Data were compared using a one-way ANOVA with a correspondingTukey's post-hoc test. (*) indicates statistically significantdifferences from control (p<0.05).

FIG. 22 demonstrates that when combined, Δ⁹-THC and CBD produced asignificant increase in the activation of multiple caspases. The effectsof cannabinoids on caspase and p8 expression were analyzed using Westernanalysis. U251 cells were treated for three days with THC (1.7 μM), CBD(0.4 μM), or a combination of Δ⁹-THC (1.7 μM) and CBD (0.4 μM). Proteinswere extracted and analyzed for cleaved caspase 3, 7, 9 and PARP. Blotsare representative of at least 3 independent experiments.

FIG. 23A-E presents that CB₂ activation and corresponding increases inoxygen radical formation are involved in the inhibitory effects of thecannabinoid combination treatment. The number of U251 cells stainingpositive for annexin (apoptosis) after 3 days treatment were measuredusing FACS analysis. Cells were treated with (A) a 4:1 combination ofTHC (1.7 μM) and CBD (0.4 μM), (B) 2.5 μM Δ⁹-THC, or (C) 2.0 μM CBD inthe presence of 0.5 μM of the CB₁ antagonist, SR141716A (“SR1”), 0.5 μMof the CB₂ antagonist, SR144528 (“SR2”) or 20 μM α-tocopherol (“TCP”).Percent control was calculated as positive annexin staining of thetreated cells minus control cells. (D) The effects of cannabinoids onpERK activity were analyzed using Western analysis. α-tubulin was usedas a loading control (LC). U251 cells were treated with vehicle or theindicated drugs for three days. (E) The production of cellular radicaloxygen species (ROS)/H₂O₂ was measured using2,7-dichlorodihydrofluorescein and FACS analysis. U251 cells weretreated with vehicle or a 4:1 combination of Δ⁹-THC (1.7 μM) and CBD(0.4 μM). Data are the mean of at least 3 independent experiments,bars±SE. Data were compared using a one-way ANOVA with a Bonferroni'smultiple comparison post-hoc analyses. (*) indicates statisticallysignificant differences from control (p<0.05). (#) indicatesstatistically significant differences from the combination treatment ofTHC/CBD (p<0.05).

FIG. 24A-D demonstrates that the O-1663 CBD analog is more potent thanCBD at inhibiting cancer cell growth and invasion, and Id-1 expression.(A) Provides the chemical structure of CBD and the O-1663 CBD analog.(B) CBD inhibition of MDA MB231 and U251 cancer cell growth as assessedby a MTT assay. (C) Cancer cells were treated with 1.0 μM of cannabinoidfor two days and then analyzed for Id-1 protein expression.Normalization was carried out by stripping the blots and re-probing witha monoclonal anti-tubulin antibody. (D) MDA-MB231 breast cancer cellswere treated with CBD (1.0 μM) or the O-1663 CBD analog (1.0 μM) for twodays and then assessed for their ability to invade through areconstituted extracellular matrix using the Boyden chamber assay; (*)indicates statistically significant difference from control (p<0.05);and (#) indicates statistically significant differences from CBD(p<0.05).

FIG. 25A-D provides a comparison of the O-1663 CBD analog and CBD forthe inhibition of breast cancer cell proliferation/viability, invasionand Id-1 expression. (A) Mouse 4T1 and (B) human MDA-MB231 breast cancercells were treated with vehicle, CBD or the O-1663 CBD analog for 2 daysalone or in the presence of α-tocopherol (“TOC”), the CB1 receptorantagonist (SR141716A “SR1”), or the CB2 receptor antagonist (SR144528“SR2”). Cell proliferation/viability was then evaluated using the MTTassay. (C) MDA-MB231 breast cancer cells were treated with CBD or theO-1663 CBD analog for 3 days alone or in the presence of TOC, SR1, orSR2. The ability of the cells to migrate and invade in modified Boydenchambers was then determined. The percentage relativeproliferation/viability and invasion were calculated as the effect ontreated cells/vehicle cells×100. Respective controls (vehicle treatedcells) were set as 100%. (D) Proteins from MDA-MB231 cells treated withvehicle (control), CBD (1.0 μM), or the O-1663 CBD analog (1.0 μM) forthree days and were extracted and analyzed for Id-1 and Id-2 (marker ofgood prognosis and specificity for targeting Id-1) immunostaining usingWestern blot analysis. Data were compared using a one-way ANOVA with acorresponding Dunnett's post-hoc test. (*), (#), (5) indicatestatistically significant differences from control, CBD and O-1663analog respectively (p<0.01).

FIG. 26A-D provides data showing that the O-1663 CBD analog targetedanti-tumor pathways unique to both CBD and THC and is more potent thanCBD at generating ROS. (A) MDA-MB231 cells were treated with vehicle,CBD, THC or O-1663 CBD analog for two days. The production of ROS wasthen measured using 2′-7′ dichloro-dihydrofluorescein and cell flowcytometry. (B) MDA-MB231 cells were treated with CBD or O-1663 CBDanalog for two days in the presence or absence of SR2. (C) MDA-MB231breast cancer cells were treated with vehicle, CBD, THC or O-1663 CBDanalog for 2 days alone. The relative change in p8 mRNA expression(stress associated gene up-stream of autophagy) was evaluated. (D)Proteins from MDA-MB231 cells treated with vehicle, CBD, THC or O-1663CBD analog for two days were extracted and analyzed for LC3immunostaining (an autophagosome/autophagy marker) using Western blotanalysis. Data are the mean of at least 3 independent experiments,bars±SE. Data were compared using a one-way ANOVA with a correspondingDunnett's post-hoc test. (*) indicates statistically significantdifferences from control (p<0.05).

FIG. 27A-E demonstrates that the O-1663 CBD analog is more potent thanCBD in inhibiting breast cancer metastasis. Lung metastases weregenerated in BALB/c mice by tail vein injection of 2×10⁴ 4T1 cells. Oneday after the injection, the tumor bearing mice were injected i.p. oncea day with vehicle, CBD or O-1663 CBD analog for 14 days. (A) Percentmetastasis (total metastastic foci in treated/vehicle×100) and (B) thenumber of lung metastatic foc±2 mm were evaluated. (C) Lung metastaseswere generated in athymic nu/nu mice after tail vein injection of 5×10⁵MDA-MB231. One day after the injection, the tumor bearing mice wereinjected i.p. once a day with vehicle, CBD or O-1663 CBD analog for sixweeks and the percent metastasis was compared. (D) Lung metastases weregenerated in BALB/c mice by tail vein injection of 2×10⁴ 4T1 cells. Oneday after the injection, the tumor bearing mice were injected i.p. oncea day with vehicle or 1 mg/kg of cannabinoids for 14 days. Percentmetastasis and (E) the number of lung metastatic foc±2 mm were compared.p<0.0001 (one-way ANOVA; *p<0.05, Tukey's post-hoc test). n.s.=notsignificant.

FIG. 28A-C provides that the O-1663 CBD analog produced robustinhibition of advanced stage breast metastasis. Lung metastases weregenerated in BALB/c mice by tail vein injection of 2×10⁴ 4T1 cells. Oneweek after the injection, the tumor bearing mice were injected i.p. oncea day with vehicle, CBD, or O-1663 CBD analog (0.5 and 1 mg/kg) for 14days. (A) Percent metastasis (total metastastic foci intreated/vehicle×100) and (B) lung metastatic foc±2 mm were evaluated.(C) Mice treated with vehicle, CBD or O-1663 CBD analog (mg/kg) startingone week after tail vein injection of 2×10⁴ 4T1 cells were observeduntil they demonstrated signs of disease progression that necessitatedeuthanasia. Survival between groups was compared using Kaplan-Meieranalysis.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a compound”includes a plurality of such compounds and reference to “the cancer”includes reference to one or more cancers, and so forth.

Also, the use of “or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising,” “include,” “includes,”“including,” “have,” “haves,” and “having” are interchangeable and notintended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of:”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the disclosed methods and compositions, the exemplarymethods, devices and materials are described herein.

For purposes of this disclosure, the term “alkyl” refers to an alkylgroup that contains 1 to 30 carbon atoms. Where if the alkyl groupcontains more than 1 carbon, the carbons may be connected in a linearmanner, or alternatively if there are more than 2 carbons then thecarbons may also be linked in a branched fashion so that the parentchain contains one or more secondary, tertiary, or quaternary carbons.An alkyl may be substituted or unsubstituted, unless stated otherwise.Specific substituted alkyl groups include haloalkyl groups, particularlytrihalomethyl groups, such as trifluoromethyl groups.

For purposes of this disclosure, the term “alkenyl” refers to an alkenylgroup that contains 1 to 30 carbon atoms. While a C₁-alkenyl can form adouble covalent bond to a carbon of a parent chain, an alkenyl group ofthree or more carbons can contain more than one double covalent bond. Itcertain instances the alkenyl group will be conjugated, in other casesan alkenyl group will not be conjugated, and yet other cases the alkenylgroup may have stretches of conjugation and stretches of nonconjugation.Additionally, if there is more than 1 carbon, the carbons may beconnected in a linear manner, or alternatively if there are more than 2carbons then the carbons may also be linked in a branched fashion sothat the parent chain contains one or more secondary, tertiary, orquaternary carbons. An alkenyl may be substituted or unsubstituted,unless stated otherwise.

For purposes of this disclosure, the term “alkynyl” refers to an alkynylgroup that contains 1 to 30 carbon atoms. While a C₁-alkynyl can form atriple covalent bond to a carbon of a parent chain, an alkynyl group ofthree or more carbons can contain more than one triple covalent bond.Where if there is more than 1 carbon, the carbons may be connected in alinear manner, or alternatively if there are more than 3 carbons thenthe carbons may also be linked in a branched fashion so that the parentchain contains one or more secondary, tertiary, or quaternary carbons.An alkynyl may be substituted or unsubstituted, unless stated otherwise.

For purposes of this disclosure, the term “cylcloalkyl” refers to analkyl that contains at least 3 carbon atoms but no more than 12 carbonatoms connected so that it forms a ring. A “cycloalkyl” for the purposesof this disclosure encompasses from 1 to 7 cycloalkyl rings, whereinwhen the cycloalkyl is greater than 1 ring, then the cycloalkyl ringsare joined so that they are linked, fused, or a combination thereof. Acycloalkyl may be substituted or unsubstituted, or in the case of morethan one cycloalkyl ring, one or more rings may be unsubstituted, one ormore rings may be substituted, or a combination thereof. A “cycloalkylgroup” can include bicyclic and tricyclic alkyl groups.

For purposes of this disclosure, the term “aryl” refers to a conjugatedplanar ring system with delocalized pi electron clouds that contain onlycarbon as ring atoms. An “aryl” for the purposes of this disclosureencompasses from 1 to 7 aryl rings wherein when the aryl is greater than1 ring the aryl rings are joined so that they are linked, fused, or acombination thereof. An aryl may be substituted or unsubstituted, or inthe case of more than one aryl ring, one or more rings may beunsubstituted, one or more rings may be substituted, or a combinationthereof. More specifically, substituted aryl groups include acetylphenylgroups, particularly 4-acetylphenyl groups; fluorophenyl groups,particularly 3-fluorophenyl and 4-fluorophenyl groups; chlorophenylgroups, particularly 3-chlorophenyl and 4-chlorophenyl groups;methylphenyl groups, particularly 4-methylphenyl groups, andmethoxyphenyl groups, particularly 4-methoxyphenyl groups.

For purposes of this disclosure, the term “heterocycle” refers to ringstructures that contain at least 1 noncarbon ring atom. A “heterocycle,”as used herein, encompasses from 1 to 7 heterocycle rings wherein whenthe heterocycle is greater than 1 ring the heterocycle rings are joinedso that they are linked, fused, or a combination thereof. A heterocyclemay be aromatic or nonaromatic, or in the case of more than oneheterocycle ring, one or more rings may be nonaromatic, one or morerings may be aromatic, or a combination thereof. A heterocycle may besubstituted or unsubstituted, or in the case of more than oneheterocycle ring one or more rings may be unsubstituted, one or morerings may be substituted, or a combination thereof. Typically, thenoncarbon ring atom is N, O, S, Si, Al, B, or P. In case where there ismore than one noncarbon ring atom, these noncarbon ring atoms can eitherbe the same element, or combination of different elements, such as N andO. Examples of heterocycles include, but are not limited to: amonocyclic heterocycle such as, aziridine, oxirane, thiirane, azetidine,oxetane, thietane, pyrrolidine, pyrroline, imidazolidine, pyrazolidine,pyrazoline, dioxolane, sulfolane 2,3-dihydrofuran, 2,5-dihydrofurantetrahydrofuran, thiophane, piperidine, 1,2,3,6-tetrahydro-pyridine,piperazine, morpholine, thiomorpholine, pyran, thiopyran,2,3-dihydropyran, tetrahydropyran, 1,4-dihydropyridine, 1,4-dioxane,1,3-dioxane, dioxane, homopiperidine, 2,3,4,7-tetrahydro-1H-azepinehomopiperazine, 1,3-dioxepane, 4,7-dihydro-1,3-dioxepin, andhexamethylene oxide; and polycyclic heterocycles such as, indole,indoline, isoindoline, quinoline, tetrahydroquinoline, isoquinoline,tetrahydroisoquinoline, 1,4-benzodioxan, coumarin, dihydrocoumarin,benzofuran, 2,3-dihydrobenzofuran, isobenzofuran, chromene, chroman,isochroman, xanthene, phenoxathiin, thianthrene, indolizine, isoindole,indazole, purine, phthalazine, naphthyridine, quinoxaline, quinazoline,cinnoline, pteridine, phenanthridine, perimidine, phenanthroline,phenazine, phenothiazine, phenoxazine, 1,2-benzisoxazole,benzothiophene, benzoxazole, benzthiazole, benzimidazole, benztriazole,thioxanthine, carbazole, carboline, acridine, pyrolizidine, andquinolizidine. In addition to the polycyclic heterocycles describedabove, heterocycle includes polycyclic heterocycles wherein the ringfusion between two or more rings includes more than one bond common toboth rings and more than two atoms common to both rings. Examples ofsuch bridged heterocycles include quinuclidine,diazabicyclo[2.2.1]heptane and 7-oxabicyclo[2.2.1]heptane.

For purposes of this disclosure, the terms “heterocyclic group”,“heterocyclic moiety”, “heterocyclic”, or “heterocyclo” used alone or asa suffix or prefix, refer to a heterocycle that has had one or morehydrogens removed therefrom.

For purposes of this disclosure, the term “hetero-” when used as aprefix, such as, hetero-alkyl, hetero-alkenyl, hetero-alkynyl, orhetero-hydrocarbon, refers to the specified hydrocarbon group having oneor more carbon atoms replaced by one or more non carbon atoms. Examplesof such non carbon atoms include, but are not limited to, N, O, S, Si,Al, B, and P. If there is more than one non carbon atom in thehetero-chain then this atom may be the same element or may be acombination of different elements, such as N and O.

For purposes of this disclosure, the term “extended mixed ring system”refers to a group that is comprised of at least 2 ring structures, butno more than 7 ring structures. An “extended mixed ring system” iscomprised of at least one ring functional group that is different fromanother ring functional group. Examples of ring groups include, but arenot limited to, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, andheterocycle. Each ring may be optionally substituted. The ringscomprising the mixed extended ring system may be joined so that they arelinked, fused, or a combination thereof.

For purposes of this disclosure, the term “unsubstituted” with respectto hydrocarbons, heterocycles, and the like, refers to structureswherein the specified group contains no substituents.

For purposes of this disclosure, the term “substituted” with respect tohydrocarbons, heterocycles, and the like, refers to structures whereinthe specified group contains one or more substituents.

For purposes of this disclosure, the term “substituent” refers to anatom or group of atoms substituted in place of a hydrogen atom. Forpurposes of this disclosure, a substituent would include deuteriumatoms.

For purposes of this disclosure, the term “functional group” or “FG”refers to specific groups of atoms attached to a parent chain or locatedwithin a parent chain that are responsible for the characteristicchemical reaction of those molecules. While the same functional groupwill undergo the same or similar chemical reaction(s) regardless of thesize of the molecule it is a part of, its relative reactivity can bemodified by nearby functional groups. The atoms of functional groups arelinked to each other and to the rest of the molecule by covalent bonds.Larger functional groups, such as hydrocarbons, esters, andheterocycles, can be optionally substituted. Examples of FG that areused in this disclosure include, but are not limited to, alkyls,alkenyls, alkynyls, aryls, hetero-alkyls, hetero-alkenyls,hetero-alkynyls, cycloalkyls, cycloalkenyls, cycloalkynyls,heterocycles, halos, hydroxyls, anhydrides, carbonyls, carboxyls,carbonates, carboxylates, aldehydes, haloformyls, esters, hydroperoxy,peroxy, ethers, orthoesters, carboxamides, amines, imines, imides,azides, azos, cyanates, isocyanates, nitrates, nitriles, isonitriles,nitrosos, nitros, nitrosooxy, pyridyls, sulfhydryls, sulfides,disulfides, sulfinyls, sulfos, thiocyanates, isothiocyanates,carbonothioyls, phosphinos, phosphonos, phosphates, silyls, and Si(OH)₃.

As used herein, a wavy line intersecting another line that is connectedto an atom indicates that this atom is covalently bonded to anotherentity that is present but not being depicted in the structure. A wavyline that does not intersect a line but is connected to an atomindicates that this atom is interacting with another atom by a bond orsome other type of identifiable association.

As used herein, a bond indicated by a straight line and a dashed lineindicates a bond that may be a single covalent bond or alternatively adouble covalent bond.

As used herein, the terms “CBD derivatives,” “CBD analogs,” “derivativesof CBD,” or “analogs of CBD” are used interchangeably and refer tocompounds that are both structurally and functionally related tocannabidiol. For example, a derivative of cannabidiol includes acompound of structural Formulas I, II, II(a), III or III(a).

The term “cannabinoids” generally refers to a group of substances thatare structurally related to Δ⁹-tetrahydrocannabinol (“THC”) or that bindto cannabinoid receptors. Plant cannabinoids are stable compounds withlow toxicity profiles that are well tolerated by animals and humansduring chronic administration. A variety of chemical classes ofcannabinoids are useful in the methods provided herein includingcannabinoids structurally related to THC, aminoalkylindoles, theeicosanoids related to the endocannabinoids, 1,5-diarylpyrazoles,quinolines and arylsulphonamides and additional compounds that do notfall into these standard classes but bind to cannabinoid receptors.

The term “pharmaceutically acceptable” as in pharmaceutically acceptablesalt or pharmaceutically acceptable counter ion, refers to compounds,salts, or ions that are tolerated by a subject for topical, or internaluse.

The term “pharmaceutically acceptable salt” refers to making a saltformation of a compound disclosed herein. Salt formation can be used asa means of varying the properties of the compounds disclosed herein, forexample, to increase or decrease solubility of the compounds, to improvestability of the compounds, to reduce toxicity of the compounds, and/orto reduce the hygroscopicity of the compounds. There are a wide range ofchemically diverse acids and bases, with a range of pKa values,molecular weights, solubilities and other properties, that can used formaking pharmaceutically acceptable salts of the compounds disclosedherein. Examples of pharmaceutically acceptable acid addition saltsinclude, but are not limited to, hydrochloride, hydrobromide,hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate,isonicotinate, acetate, lactate, salicylate, citrate, tartrate,pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate,fumarate, gluconate, glucaronate, saccharate, formate, benzoate,glutamate, methanesulfonate, ethanesulfonate, benzensulfonate,p-toluenesulfonate and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Certain compounds ofthe disclosure can form pharmaceutically acceptable salts with variousamino acids. Examples of pharmaceutically acceptable base addition saltsinclude, but are not limited to, aluminum, calcium, lithium, magnesium,potassium, sodium, zinc, and diethanolamine salts. For additionalexamples of pharmaceutical salts that can used to practice thisdisclosure, see P. H. Stahl and C. G. Wermuth (eds.), PharmaceuticalSalts Properties, Selection, and Use (2d ed. 2011) Wiley and SonsPublisher, ISBN: 978-3-90639-051-2.

The term “pharmaceutically acceptable counter ion” either refers topharmaceutically acceptable cations including, but not limited to,alkali metal cations (e.g., Li⁺, Na⁺, K⁺), alkaline earth metal cations(e.g., Ca²⁺, Mg²⁺), non-toxic heavy metal cations and ammonium (NH₄ ⁺)and substituted ammonium (N(R′)₄ ⁺, where R′ is hydrogen, alkyl, orsubstituted alkyls, i.e., including, methyl, ethyl, or hydroxyethyl,specifically, trimethyl ammonium, triethyl ammonium, and triethanolammonium cations); or pharmaceutically-acceptable anions including, butnot limited to, halides (e.g., Cl⁻, Br⁻), sulfate, acetates (e.g.,acetate, trifluoroacetate), ascorbates, aspartates, benzoates, citrates,and lactate.

The term “metastasis” generally refers to a multi-step process by whichaggressive cancer cells spread out of the primary tissue and into othertissues of the body. Aggressive cancer cells that are nourished throughangiogenesis, can migrate out of the primary tissue, and invade into theblood stream. These migratory aggressive cancer cells can remain vitalby escaping the immune response, and consequently evade the blood streamand invade other tissues of the body. These cells can then proliferateto create secondary tumors.

A “cell proliferative disorder” is any cellular disorder in which thecells proliferate more rapidly than normal tissue growth. Thus a“proliferating cell” is a cell that is proliferating more rapidly thannormal cells. A proliferative disorder can include but is not limited toneoplasms.

A “neoplasm” refers to an abnormal tissue growth, generally forming adistinct mass that grows by cellular proliferation more rapidly thannormal tissue growth. Neoplasms show partial or total lack of structuralorganization and functional coordination with normal tissue. These canbe broadly classified into three major types. Malignant neoplasmsarising from epithelial structures are called carcinomas, malignantneoplasms that originate from connective tissues such as muscle,cartilage, fat or bone are called sarcomas and malignant tumorsaffecting hematopoetic structures (structures pertaining to theformation of blood cells) including components of the immune system, arecalled leukemias and lymphomas. Unless stated otherwise, a “neoplasm” asused herein refers to all types of neoplasms. A tumor is the neoplasticgrowth of the disease cancer. As used herein, a neoplasm, also referredto as a “tumor”, encompasses hematopoietic neoplasms as well as solidneoplasms. Other neoplasm based disorders include, but are not limitedto neurofibromatosis, melanoma, breast cancers, head and neck cancers(e.g., brain cancers such as glioblastoma multiforme), gastrointestinalcancers and the like. A cancer generally refers to any neoplasticdisorder, including such cellular disorders as, for example, braincancer, glioblastoma multiforme (GBM), renal cell cancer, Kaposi'ssarcoma, chronic leukemia, breast cancer, sarcoma, ovarian carcinoma,rectal cancer, throat cancer, melanoma, colon cancer, bladder cancer,mastocytoma, lung cancer and gastrointestinal or stomach cancer.

A “subject” generally refers to mammals such as human patients andnon-human primates, as well as experimental animals such as rabbits,rats, and mice, and other animals. Animals include all vertebrates,e.g., mammals and non-mammals, such as sheep, dogs, cows, chickens,amphibians, and reptiles.

All publications mentioned herein are incorporated herein by referencein full for the purpose of describing and disclosing the methodologies,which are described in the publications, which might be used inconnection with the description herein. The publications discussed aboveand throughout the text are provided solely for their disclosure priorto the filing date of the present application. Nothing herein is to beconstrued as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior disclosure. Moreover, withrespect to similar or identical terms found in the incorporatedreferences and terms expressly defined in this disclosure, the termdefinitions provided in this disclosure will control in all respects.

Activation of the two cannabinoid receptors, cannabinoid receptor 1(“CB₁”) and cannabinoid receptor 2 (“CB₂”), can lead to the inhibitionof cell proliferation and induction of apoptosis in multiple types ofcancer cell lines resulting in the reduction of tumor growth in vivo.The CB₁ and CB₂ receptors are members of the G-protein coupled receptor(GPCR) superfamily, and can interact with five structurally distinctclasses of compounds. These include the plant-derived classicalcannabinoids, such as THC and CBN; the non-classical bicycliccannabinoid agonists, such as CP55, 940; the endogenous cannabinoidagonists, such as anandamide (AEA); and the aminoalkylindole (AAI)agonists, such as WIN55, 212-2; and the antagonist/inverse agonists,such as SR141716A.

Interaction sites, independent of CB₁ and CB₂ receptors, may also beresponsible for the anticancer activity of cannabinoids. There are morethan 60 cannabinoids in Cannabis sativa. In addition to THC, thecompounds cannabidiol (“CBD”), cannabinol (“CBN”), and cannabigerol(“CBG”) are also present in reasonable quantities. CBN has low affinityfor CB₁ and CB₂ receptors, whereas the non-psychotropic cannabinoids,CBD and CBG, have negligible affinity for the cloned receptors.

The studies presented herein demonstrated that the helix-loop-helixprotein Id-1, an inhibitor of basic helix-loop-helix (bHLH)transcription factors, plays a crucial role during breast cancerprogression. Id-1 stimulates proliferation, migration and invasion inbreast cancer cells. Moreover, targeting Id-1 expression partially inbreast cancer cells reduces invasion and breast cancer metastasis invitro and in preclinical animal models. The disclosure provides thatId-1 is a therapeutic target for treating disorders and diseasesassociated with Id polynucleotide expression. The disclosure furtherprovides that Id-associated cell proliferative disorders, such as breastcancer, can be treated by inhibiting Id-1 expression and/or activity.This approach may be highly effective and safe in advanced breast cancerpatients, given (1) the relationship between high Id-1 expression levelsand aggressive breast cancer cell behaviors; (2) partial reduction inId-1 activity can achieve significant outcomes; and (3) Id-1 expressionis low in normal adult tissues, thereby eliminating unwanted toxicitiesgenerally associated with currently available therapeutic modalities.

Id-1 protein plays a key role in the malignant progression of manyaggressive and invasive human cancers such as: leukemia, melanoma,hepatocellular carcinoma, colorectal adenocarcinoma, pancreatic cancer,lung cancer, kidney cancer, medullary thyroid cancer, papillary thyroidcancer, astrocytic tumor, neuroblastoma, Ewing's sarcoma, ovarian tumor,cervical cancer, endometrial carcinoma, breast cancer, prostate cancer,malignant seminoma, and squamous cell carcinomas, such as esophagealcancer, and head and neck cancer. Accordingly, Id-1 associated cellproliferative disorders include, but are not limited to, leukemias,melanomas, squamous cell carcinomas (SCC) (e.g., head and neck,esophageal, and oral cavity), hepatocellular carcinomas, colorectaladenocarcinomas, pancreatic cancers, lung cancers, kidney cancers,medullary thyroid cancers, papillary thyroid cancers, astrocytic tumors,neuroblastomas, Ewing's sarcomas, ovarian tumors, cervical cancers,endometrial carcinomas, breast cancers, prostate cancers, and malignantseminomas.

Approaches for targeting Id-1 expression include gene therapy usingantisense oligonucleotide, siRNA, non-viral or viral plasmid-basedstrategies. In addition, the development of new strategies to modulateId-1 expression/functional activity includes the identification of smallmolecules that modulate the activity of Id-1. A range of small moleculesthat target the molecular pathology of cancer are now being developed,and a significant number of them are being tested in ongoing humanclinical trials.

The disclosure demonstrates that the compounds disclosed herein areinhibitors of Id-1 expression and modulate other Id-helix-loop-helixprotein expression, such as Id-2. The use of the compounds disclosedherein, therefore, represents a novel strategy for the treatment ofcancer.

Metastasis is the final and often fatal step in the progression ofaggressive cancers. Currently available therapeutic strategies at thisstage of cancer progression are often non-specific, have only marginalefficacy and are highly toxic. This is in part due to the lack ofknowledge about the molecular mechanisms regulating the development ofaggressive cancers. Therapeutic approaches targeting only specificmechanisms involved in the development of aggressive cancers are inurgent need. The expectation would be that this strategy would reduceunwanted toxicities associated with the therapy itself.

The compounds disclosed herein were found to down-regulate Id-1expression in metastatic foci of the lung and corresponding breastcancer metastasis in mice. Moreover, the O-1663 CBD analog was found tobe unexpectedly more potent than CBD and other compounds at inhibitingbreast cancer metastasis in a mouse model where tumor cellaggressiveness is highly dependent on the expression of Id-1. Inaddition, data provided herein indicates that the compounds disclosedherein were surprisingly effective in inhibiting cell proliferation andinvasiveness, and could induce cell death in-vitro and in-vivo.

Human GBMs are highly heterogeneous and vary in their response totherapeutic treatments. As described herein, this heterogeneity isreflected in the response of multiple aggressive GBM cancers cell linesto the anti-proliferative activity of synthetic and naturally occurringcannabinoids. The disclosure also provides for other constituents ofmarijuana, such as THC, that inhibit GBM cell growth and inducesapoptosis. The disclosure demonstrates that the addition of CBD orCBD-based analogs to THC improves the overall potency and efficacy ofTHC in the treatment of cancer.

The compounds disclosed herein were shown to inhibit Id-1 expression andtumor progression in a mouse model of brain cancer. In particular, theO-1663 CBD analog was found to be particularly effective in inhibitingId-1 and corresponding cell proliferation and invasion by breast cancercells and other aggressive cancerous cells.

Compositions comprising compounds disclosed herein were found tomodulate Id-1 and Id-2 expression in tested cancer cell lines. Moreover,as Id-1 expression was found to be up-regulated during the progressionof almost all types of solid tumors investigated, compositionscomprising compounds disclosed herein can provide a generalizedtherapeutic strategy for the treatment of various aggressive cancers.Accordingly, provided herein are methods for modulating the activity ofa metastatic cell by regulating the activity of a target Id polypeptideby using a compound disclosed herein. For the purposes of thisdisclosure, “regulating the activity of a target Id polypeptide” canalso include: (1) mechanisms for modulating endogenous nucleic acidsequences which encode a target Id protein so that Id polypeptide levelsare decreased in a cell; (2) introducing exogenous nucleic acidsequences that inhibit Id mRNA and/or protein expression in a cell; and(3) increasing the turnover rate of endogenous Id polypeptides such thatId polypeptide levels are decreased in a cell.

In a particular embodiment, the disclosure provides methods that can beused to identify substances that modulate the biological activity of anId polypeptide, such as by modulating the expression of an Id nucleicacid sequence which encodes an Id polypeptide. In another embodiment, amethod disclosed herein can be used to identify one or more compoundsthat bind to Id regulatory sequences. In yet another embodiment, amethod disclosed herein can be used to identify one or more substanceswhich modulate the biological activity of Id polypeptide by affectingthe half-life of an Id polypeptide.

In a particular embodiment, the disclosure provides methods for treatingcell proliferation disorders by administering one or more compoundsdisclosed herein. In general, these methods can be used to treatdisorders related to neoplastic cells and the metastasis thereof. In acertain embodiment, the disclosure provides methods for treating cellproliferation disorders by administering one or more compounds disclosedherein which regulate the expression and/or activity of endogenous Idpolypeptides and/or the half-life of endogenous Id polypeptides. In afurther embodiment, the disclosure provides methods for treatingdisorders that can be ameliorated by modulating one or more Idpolypeptides expression.

The disclosure also pertains to the field of predictive medicine inwhich diagnostic assays, prognostic assays, pharmacogenomics, andmonitoring clinical trails are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. As such, the disclosurecontemplates use of methods provided herein to screen, diagnose, stage,prevent and/or treat disorders characterized by expression orover-expression of an Id helix-loop-helix polypeptide, such as Id-1.Accordingly, a subject can be screened to determine the level of aparticular Id's expression or activity. A subject with a cellproliferative disorder can also be screened to determine whetherabnormally proliferating cells would be susceptible to techniquesdisclosed herein, including inhibiting the expression or over expressionof an Id polypeptide.

The disclosure also provides for prognostic (or predictive) assays fordetermining whether an individual is at risk of developing a disorderassociated with elevated/reduced expression of a target Id polypeptide.Such assays can be used for prognostic or predictive purpose to therebyprophylactically treat an individual prior to the onset of a disordercharacterized by or associated with over expression or activity of atarget Id polypeptide, such as Id-1.

As described herein the compounds disclosed herein are significantlymore effective than other cannabinoid compounds at inhibiting theexpression of genes and proteins that modulate cancer aggressiveness(e.g. Id-1) in, for example, breast cancer aggressiveness. The data alsoindicate that Id-1 is a key factor for breast cancer cellaggressiveness. The down-regulation of gene expression of Id-1 resultedfrom inhibiting the endogenous Id-1 promoter. As shown in the Figurespresented herein, CBD and the 0-1663 CBD analog effectively inhibits theexpression of Id-1 in metastatic breast cancer cells and GBM cells.

To determine general structural components of CBD that were responsiblefor its inhibitory activity, CBD was compared against structurallyrelated cannabinoid compounds for their ability to inhibit Id-1 (e.g.,see FIG. 1). Δ⁹-Tetrahydrocannabinol (“THC”) had no activity againstId-1. THC is structurally related to CBD with the primary exceptionbeing that the B ring or 1,1′-di-methyl-pyrane ring (e.g. see FIG. 1,panel E) of THC has been opened in CBD. CP55, 940 was a compound thatinhibited Id-1 expression, however, it was still less effective thanCBD.

As presented herein, cannabinoids CBD and THC are effective atinhibiting breast cancer progression. However, they each target uniquepathways. In a mouse model of metastasis, a detailed pharmacologicalassessment of the non-toxic agent, CBD, revealed the drugs ability tosignificantly extend survival. This effect was found to be directlyrelated to down-regulation of Id-1 expression in vivo. In addition, itwas found that CBD could reduce the number and size of metastatic fociin advanced stages of breast cancer metastasis. Treatment with CBD inadvanced stage models of metastasis also resulted in moderate increasesin survival. In hopes of generating more potent and efficacioustherapeutic compounds for treating cell proliferative disorders, analogsbased on the CBD structure were screened for inhibiting breast cancerviability/proliferation, and invasion and Id-1 expression. These CBDbased analogs were also screened to see if they were active at targetingCB₂ receptors. The analogs disclosed herein were developed so as to havedesirable properties from both THC and CBD, achieving a synergy notpossible by administering THC or CBS alone. By dual targeting eachunique antitumor pathway with a single compound, results in a robustsynergistic inhibition of advanced stages of metastasis without thedrawbacks and side effects of administering each compound individually.

Based upon initial screening assays, a CBD analog was found that wasmore potent than CBD in inhibiting Id-1 expression, and retarded cellproliferation, metastasis, and invasion by various cancer cell lines(e.g., see FIGS. 24-30). This analog, termed 0-1663, is a cannabidiolderivative which has 2 linked unsubstituted cyclohexyl rings instead ofthe substituted cyclohexylalkenyl ring of cannabidiol. Moreover, theO-1663 CBD analog contains an aliphatic C₅ alkyl group which contains1,1-dimethyl substitutions. The data provided herein show that theunique activity (Id-1 inhibition) of the O-1663 CBD analog can beattributed in-part to hydrophobic cycloalkyl rings and the possession ofan extended substituted alkyl side chain. The results from the O-1663CBD analog provide a basis for generating additional compounds usingrational drug design so as to generate even more potent and efficacioustherapeutics.

In a particular embodiment, the disclosure provides for a compoundhaving the structure of Formula I:

or a pharmaceutically acceptable salt, or prodrug thereof, wherein:

R¹ is selected from the group comprising an optionally substituted(C₅-C₁₂)alkyl, an optionally substituted hetero-(C₅-C₁₂)alkyl, anoptionally substituted (C₅-C₁₂)alkenyl, an optionally substitutedhetero-(C₅-C₁₂)alkenyl, an optionally substituted (C₅-C₁₂)alkynyl, anoptionally substituted hetero-(C₅-C₁₂)alkynyl, an optionally substituted(C₅-C₁₂)cycloalkyl, an optionally substituted (C₅-C₁₂) cycloalkenyl, anoptionally substituted (C₅-C₁₂) cycloalkynyl, an optionally substituted(C₄-C₁₁)heterocycle, an optionally substituted aryl, and an optionallysubstituted extended mixed ring system;

R²-R³ are each independently selected from the group comprisinghydroxyl, (C₁-C₂)alkoxy, carboxylic acid, amine, halo, cyano, or(C₁-C₃)ester;

R⁴-R⁵ are each independently selected from the group comprisinghydrogen, hydroxyl, (C₁-C₂)alkoxy, carboxylic acid, amine, halo, cyano,and ester;

R⁶ is selected from the group comprising an optionally substituted(C₁-C₁₂)alkyl, an optionally substituted hetero(C₁-C₁₁)alkyl, anoptionally substituted (C₁-C₁₂)alkenyl, an optionally substitutedhetero(C₁-C₁₁)alkenyl, an optionally substituted (C₁-C₁₂)alkynyl, and anoptionally substituted hetero(C₁-C₁₁)alkynyl.

In a certain embodiment, in the case of a compound having a structure ofFormula I:

where R² and R³ are hydroxyls, R⁶ is an alkyl, or substituted alkyl ofat least 6 carbon atoms, and R¹ is a cyclohexylalkenyl with the generalstructure of

then Y is not selected from the group consisting of a H, —OH, aryl,substituted aryl, alkyl, substituted alkyl, carboxyl, aminocarbonyl,alkylsulfonylaminocarboxyl, and alkoxycarbonyl.

In another embodiment, in the case of a compound having structure ofFormula I:

where R⁶ is a 1,1-dimethyl-heptyl group, R² and R³ are hydroxyls, and R⁴and R⁵ are hydrogens, then R¹ is not selected from the group consistingof:

In a certain embodiment, in the case of a compound having structure ofFormula I:

where R⁶ is a 1,1-dimethyl-heptyl group, R² and R³ are methoxys, and R⁴and R⁵ are hydrogens, then R¹ is not selected from the group consistingof:

In a certain embodiment, in the case of a compound having a structure ofFormula I:

where R⁶ is a methyl, n-propyl group, n-butyl group, or n-pentyl group,R² is a hydroxyl, R³ is a hydroxyl or methoxy, R⁴ is hydrogen orcarboxylic acid, and R⁵ is hydrogen, then R¹ is not selected from thegroup consisting of:

In another embodiment, in the case of a compound having a structure ofFormula I:

where R⁶ is a 1,1-dimethyl-butyl group, R² and R³ are hydroxyls, and R⁴and R⁵ are hydrogen, then R¹ is not

In another embodiment, in the case of a compound having a structure ofFormula I:

where R⁶ is a methyl group, R² and R³ are methoxys, and R⁴ and R⁵ arehydrogen, then R¹ is not

In a further embodiment, the disclosure provides for a compound having astructure of Formula I, wherein R¹ is selected from the groupcomprising:

and wherein R²⁰ is selected from the group comprising hydrogen,deuterium, FG, optionally substituted (C₅-C₁₂)alkyl, optionallysubstituted hetero-(C₅-C₁₂) alkyl, optionally substituted (C₅-C₁₂)alkenyl, optionally substituted hetero-(C₅-C₁₂)alkenyl, optionallysubstituted(C₅-C₁₂)alkynyl, optionally substitutedhetero-(C₅-C₁₂)alkynyl, optionally substituted (C₅-C₁₂)cycloalkyl,optionally substituted (C₅-C₁₂)cycloalkenyl, optionally substituted(C₅-C₁₂)cycloalkynyl, optionally substituted (C₄-C₁₁)heterocycle,optionally substituted aryl, or optionally substituted extended mixedring system.

In a certain embodiment, the disclosure provides for a compound ofFormula I, having the structure of Formula II:

or a pharmaceutically acceptable salt, or prodrug thereof, wherein:

X is independently C or N;

R²-R³ are each independently selected from the group comprisinghydroxyl, (C₁-C₂)alkoxy, carboxylic acid, amine, halo, cyano, and(C₁-C₃) ester;

R⁴-R⁵ are each independently selected from the group comprisinghydrogen, deuterium, hydroxyl, (C₁-C₂)alkoxy, carboxylic acid, amine,halo, cyano, and (C₁-C₃) ester;

R⁶ is selected from the group comprising unsubstituted (C₁-C₁₂) alkyl,unsubstituted hetero (C₁-C₁₁) alkyl, unsubstituted (C₁-C₁₂) alkenyl,unsubstituted hetero (C₁-C₁₁) alkenyl, unsubstituted (C₁-C₁₂) alkynyl,and unsubstituted hetero (C₁-C₁₁) alkynyl;

R¹⁰-R¹⁹ are each independently selected from the group comprisinghydrogen, deuterium, FG, optionally substituted (C₁-C₈)alkyl, optionallysubstituted hetero(C₁-C₈)alkyl, optionally substituted (C₁-C₈)alkenyl,optionally substituted hetero(C₁-C₈)alkenyl, optionally substituted(C₁-C₈)alkynyl, optionally substituted hetero(C₁-C₈)alkynyl, optionallysubstituted (C₅-C₈)cycloalkyl, optionally substituted(C₅-C₈)cycloalkenyl, optionally substituted (C₅-C₈)cycloalkynyl,optionally substituted (C₄-C₈)heterocycle, optionally substitutedextended mixed ring system; and

R²⁰ is selected from the group comprising an optionally substituted(C₅-C₁₂)cycloalkyl, an optionally substituted (C₅-C₁₂)cycloalkenyl, anoptionally substituted (C₅-C₁₂)cycloalkynyl, an optionally substituted(C₄-C₁₁)heterocycle, a substituted aryl, an optionally substituted arylhaving more than two rings, and an optionally substituted extended mixedring system.

In another embodiment, the disclosure provides a compound of Formula II,wherein R²⁰ is an optionally substituted (C₅-C₇)cycloalkyl, anoptionally substituted (C₅-C₇)cycloalkenyl, a substituted aryl, anoptionally substituted aryl of two or more rings, an optionallysubstituted heterocycle, wherein the heterocycle contains 4, 5, or 6ring atoms.

In yet another embodiment, the disclosure provides a compound of FormulaII, wherein R²⁰ is an optionally substituted heterocycle containing 4,5, or 6 ring atoms selected from the group comprising:

In yet another embodiment, the disclosure provides a compound of FormulaII, wherein R²⁰ is a substituted aryl or an optionally substituted arylhaving 2 or more rings selected from the group comprising:

In a particular embodiment, the disclosure provides a compound ofFormula II, wherein R²⁰ is an optionally substituted (C₅-C₇)cycloalkylring selected from the group comprising:

In another embodiment, the disclosure provides for a compound of FormulaI, having the structure of Formula II(a):

or a pharmaceutically acceptable salt, or prodrug thereof, wherein:

X is independently C or N;

R²-R³ are each independently selected from the group comprisinghydroxyl, (C₁-C₂)alkoxy, carboxylic acid, amine, halo, cyano, and(C₁-C₃) ester;

R⁴-R⁵ are each independently selected from the group comprisinghydrogen, deuterium, hydroxyl, (C₁-C₂)alkoxy, carboxylic acid, amine,halo, cyano, and (C₁-C₃) ester;

R⁷-R⁸ areeach independently selected from the group comprising (C₁-C₃)alkyl, hetero (C₁-C₃) alkyl, (C₁-C₃) alkenyl, hetero (C₁-C₃) alkenyl,(C₁-C₃)alkynyl, cyano, hydroxyl, halo, amine, ketal, hemiketal, andhetero (C₁-C₃) alkynyl;

R⁹ is selected from the group comprising optionally substituted (C₁-C₈)alkyl, optionally substituted hetero (C₁-C₈) alkyl, optionallysubstituted (C₁-C₈)alkenyl, optionally substituted hetero(C₁-C₈)alkenyl,optionally substituted (C₁-C₈)alkynyl, and optionally substituted hetero(C₁-C₈) alkynyl;

R¹⁰-R¹⁹ are each independently selected from the group comprisinghydrogen, deuterium, FG, optionally substituted (C₁-C₈)alkyl, optionallysubstituted hetero(C₁-C₈)alkyl, optionally substituted (C₁-C₈)alkenyl,optionally substituted hetero(C₁-C₈)alkenyl, optionally substituted(C₁-C₈)alkynyl, optionally substituted hetero(C₁-C₈)alkynyl, optionallysubstituted (C₅-C₈)cycloalkyl, optionally substituted(C₅-C₈)cycloalkenyl, optionally substituted (C₅-C₈)cycloalkynyl,optionally substituted (C₄-C₈)heterocycle, a substituted aryl, anoptionally substituted aryl of two or more rings, and optionallysubstituted extended mixed ring system; and

R²⁰ is selected from the group comprising a hydrogen, a deuterium, a FG,an optionally substituted (C₅-C₁₂)alkyl, an optionally substitutedhetero-(C₅-C₁₂)alkyl, an optionally substituted (C₅-C₁₂)alkenyl, anoptionally substituted hetero-(C₅-C₁₂)alkenyl, an optionally substituted(C₅-C₁₂)alkynyl, an optionally substituted hetero-(C₅-C₁₂)alkynyl, anoptionally substituted (C₅-C₁₂)cycloalkyl, an optionally substituted(C₅-C₁₂)cycloalkenyl, an optionally substituted (C₅-C₁₂)cycloalkynyl, anoptionally substituted (C₄-C₁₁)heterocycle, a substituted aryl, anoptionally substituted aryl having more than two rings, and anoptionally substituted extended mixed ring system.

In a particular embodiment, the disclosure provides for a compound ofFormula I or Formula II, having the structure of Formula III:

or a pharmaceutically acceptable salt, or prodrug thereof, wherein:

X is independently either a C or N;

R²-R³ are each independently a hydroxyl or (C₁-C₂)alkoxy;

R⁶ is selected from the group comprising an unsubstituted (C₄-C₁₂)alkyl, an unsubstituted hetero (C₁-C₁₁) alkyl, an unsubstituted(C₁-C₁₂)alkenyl, an unsubstituted hetero(C₄-C₁₁)alkenyl, anunsubstituted (C₁-C₁₂) alkynyl, and an unsubstituted hetero (C₁-C₁₁)alkynyl;

R¹¹-R¹⁸ are each independently selected from the group comprisinghydrogen, deuterium, FG, optionally substituted (C₁-C₈)alkyl, optionallysubstituted hetero(C₁-C₈)alkyl, optionally substituted (C₁-C₈)alkenyl,optionally substituted hetero(C₁-C₈)alkenyl, optionally substituted(C₁-C₈)alkynyl, and optionally substituted hetero (C₁-C₈) alkynyl; and

R²¹-R³¹ are each independently selected from the group comprisinghydrogen, deuterium, FG, optionally substituted (C₁-C₈)alkyl, optionallysubstituted hetero(C₁-C₈)alkyl, optionally substituted (C₁-C₈)alkenyl,optionally substituted hetero(C₁-C₈)alkenyl, optionally substituted(C₁-C₈)alkynyl, optionally substituted hetero(C₁-C₈)alkynyl, optionallysubstituted (C₅-C₈)cycloalkyl, optionally substituted(C₅-C₈)cycloalkenyl, optionally substituted (C₅-C₈)cycloalkynyl,optionally substituted (C₄-C₈)heterocycle, optionally substituted aryl,and optionally substituted extended mixed ring system.

In a certain embodiment, the disclosure provides for a compound ofFormula I or Formula II, having the structure of Formula III:

or a pharmaceutically acceptable salt, or prodrug thereof, wherein:

X is independently either a C or N;

R²-R³ are each independently selected from the group comprisinghydroxyl, (C₁-C₂)alkoxy, carboxylic acid, amine, halo, cyano, or(C₁-C₃)ester;

R⁴-R⁵ are each independently selected from the group comprisinghydrogen, deuterium, hydroxyl, (C₁-C₂)alkoxy, carboxylic acid, amine,halo, cyano, and (C₁-C₃)ester;

R⁷-R⁸ are each independently selected from the group comprisingoptionally substituted (C₁-C₃)alkyl, optionally substitutedhetero(C₁-C₃)alkyl, optionally substituted (C₁-C₃)alkenyl, optionallysubstituted hetero(C₁-C₃)alkenyl, optionally substituted (C₁-C₃)alkynyl,cyano, hydroxyl, halo, amine, ketal, hemiketal, and hetero (C₁-C₃)alkynyl;

R⁹ is selected from the group comprising an optionally substituted(C₁-C₈)alkyl, an optionally substituted hetero(C₁-C₈)alkyl, anoptionally substituted (C₁-C₈)alkenyl, an optionally substitutedhetero(C₁-C₈)alkenyl, an optionally substituted (C₁-C₈)alkynyl, and anoptionally substituted heteroalkynyl;

R¹⁰-R¹⁹ are each independently selected from the group comprisinghydrogen, deuterium, FG, optionally substituted (C₁-C₈)alkyl, optionallysubstituted hetero(C₁-C₈)alkyl, optionally substituted (C₁-C₈)alkenyl,optionally substituted hetero(C₁-C₈)alkenyl, optionally substituted(C₁-C₈)alkynyl, optionally substituted hetero(C₁-C₈)alkynyl, optionallysubstituted (C₅-C₈)cycloalkyl, optionally substituted(C₅-C₈)cycloalkenyl, optionally substituted (C₅-C₈)cycloalkynyl,optionally substituted (C₄-C₈)heterocycle, optionally substituted aryl,and optionally substituted extended mixed ring system; and

R²¹-R³¹ are each independently selected from the group comprisinghydrogen, deuterium, FG, optionally substituted (C₁-C₈)alkyl, optionallysubstituted hetero(C₁-C₈)alkyl, optionally substituted (C₁-C₈)alkenyl,optionally substituted hetero(C₁-C₈)alkenyl, optionally substituted(C₁-C₈)alkynyl, optionally substituted hetero(C₁-C₈)alkynyl, optionallysubstituted (C₅-C₈)cycloalkyl, optionally substituted(C₅-C₈)cycloalkenyl, optionally substituted (C₅-C₈)cycloalkynyl,optionally substituted (C₄-C₈)heterocycle, optionally substituted aryl,and optionally substituted extended mixed ring system.

In a particular embodiment, the disclosure provides for a compound ofFormula I, II or III, wherein R⁶ is selected from the group comprisingmethyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl,isopropyl, sec-butyl, (1-methyl)butyl, (1-methyl)pentyl,(1-methyl)hexyl, (1-methyl)heptyl, (1,1-dimethyl)propyl,(1,1-dimethyl)butyl, (1,1-dimethyl)pentyl, (1,1-dimethyl)hexyl,(1,1-dimethyl)heptyl, (1,2-dimethyl)propyl, (1,2-dimethyl)butyl,(1,2-dimethyl)pentyl, (1,2-dimethyl)hexyl, (1,2-dimethyl)heptyl,(1,3-dimethyl)butyl, (1,3-dimethyl)pentyl, (1,3-dimethyl)hexyl,(1,3-dimethyl)heptyl, (1,4-dimethyl)pentyl, (1,4-dimethyl)hexyl,(1,4-dimethyl)heptyl, (1,5-dimethyl)hexyl, (1,5-dimethyl)heptyl,(1,6-dimethyl)heptyl, (1,2-diethyl)butyl, (1,2-diethyl)pentyl,(1,2-diethyl)hexyl, (1,2-diethyl)heptyl, (1,2-diethyl)pentyl,(1,3-diethyl)pentyl, (1,3-diethyl)hexyl, (1,3-diethyl)heptyl,(1,4-diethyl)pentyl, (1,4-diethyl)hexyl, (1,4-diethyl)heptyl,(1,5-diethyl)hexyl, (1,5-diethyl)heptyl, (1,6-diethyl)heptyl,(1,2,3-trimethyl)butyl, (1,1,2-trimethyl)butyl, (1,1,3-trimethyl)butyl,(1,2,3-trimethyl)pentyl, (1,1,2-trimethyl)pentyl,(1,1,3-trimethyl)pentyl, (1,2,4-trimethyl)pentyl,(1,3,4-trimethyl)pentyl, (1,1,4-trimethyl)pentyl,(1,2,3-trimethyl)hexyl, (1,1,2-trimethyl)hexyl, (1,1,3-trimethyl)hexyl,(1,2,4-trimethyl)hexyl, (1,2,5-trimethyl)hexyl, (1,1,4-trimethyl)hexyl,(2,3,4-trimethyl)hexyl, (2,3,5-trimethyl)hexyl, (1,1,5-trimethyl)hexyl,(1,2,3-trimethyl)heptyl, (1,1,2-trimethyl)heptyl,(1,1,3-trimethyl)heptyl, (1,2,4-trimethyl)heptyl,(1,1,5-trimethyl)heptyl, (1,1,6-trimethyl)heptyl,(1,2,5-trimethyl)heptyl, (1,2,6-trimethyl)heptyl,(2,3,4-trimethyl)heptyl, (2,3,5-trimethyl)heptyl,(2,3,6-trimethyl)heptyl, (2,4,5-trimethyl)heptyl,(2,4,6-trimethyl)heptyl, (3,4,5-trimethyl)heptyl,(3,4,6-trimethyl)heptyl, and (4,5,6-trimethyl)heptyl.

In another embodiment, the disclosure provides for a compound of FormulaI, II, II(a), III, or III(a), selected from the group comprising:

In a further embodiment, the disclosure provides for a compound ofFormula I, II, II(a), III, or III(a), having the structure of:

The compounds disclosed herein can be prepared by methods known to oneof skill in the art and routine modifications thereof, and/or followingprocedures and schemes presented herein, and routine modificationsthereof, and/or procedures found in international applicationPCT/US2002/19569, Mahadevan et al., J. Med. Chem. 2000, 43(2):3778-86;Ben-Shabat et al., J. Med. Chem. 2006, 49(3):1113-117; Wiley et al.,JPET 2002, 301(2):679-689; Thompson et al., Synthesis 2005 4:547-550;Razdan, Rajik, The total synthesis of natural products 4, 1981:186-262,and references cited therein and routine modifications thereof.

In a particular embodiment, Scheme I or modifications thereof can beused to make one or more compounds of the disclosure.

Phenol 1 is reacted with a carbocation comprising R⁶, formed by reactingan alkyl, heteroalkyl, an alkenyl, a hetero-alkenyl, an alkynyl, or aheteroalkynyl containing a terminal tertiary hydroxyl group in thepresence of a strong acid, such as methanesulfonic acid, at an elevatedtemperature to afford compound 2. Compound 2 is reacted withbis(pinacolato)diboron in the presence of a palladium catalyst, at anelevated temperature in an appropriate solvent system, such as a mixtureof N,N-diethylethanamine and dioxane, to form a boronate esterintermediate, which is then reacted with a copper(I)halide, such ascopper(I)bromide, in an appropriate solvent system, such as a mixture ofmethanol and water, at an elevated temperature to form aryl halide 3(wherein Y is I, Br or Cl). Aryl halide 3 is then transmetallated byadding magnesium turnings in ether to form a Grignard reagent, which isthen reacted with ketone 4 to afford compound 5. Compound 5 can then bereacted with any number regents to convert, substitute, or eliminate thehydroxyl group to give compound 6.

In a certain embodiment, Scheme II or modifications thereof can be usedto make one or more compounds of the disclosure.

Benzaldehyde 7 is reacted with a Wittig reagent comprising R⁶. Theresulting alkene can be selectively reduced by using mild reducingagents to afford compound 2.

In another embodiment, Scheme III or modifications thereof can be usedto make one or more compounds of the disclosure.

Aniline 8 is reacted with a carbocation comprising R⁶, formed byreacting an alkyl, heteroalkyl, an alkenyl, an hetero-alkenyl, analkynyl, or an heteroalkynyl containing a terminal tertiary hydroxylgroup in the presence of a strong acid, such as methanesulfonic acid, atan elevated temperature to afford compound 9. Compound 9 is reacted withcompound 10 (wherein Y is a halide or good leaving group) in thepresence of a base, such as anhydrous potassium carbonate, in anappropriate solvent, such as diglyme, at an elevated temperature toafford compound 11.

In yet another embodiment, Scheme IV or modifications thereof can beused to make one or more compounds of the disclosure.

Benzaldehyde 12 is reacted with a Wittig reagent comprising R⁶. Theresulting alkene can be selectively reduced by using mild reducingagents to afford compound 9.

In a particular embodiment, Scheme V or modifications thereof can beused to make one or more compounds of the disclosure.

Compound 8 is reacted with compound 10 (wherein Y is a halide or goodleaving group) in the presence of a base, such as anhydrous potassiumcarbonate, in an appropriate solvent, such as diglyme, at an elevatedtemperature to afford compound 13. Compound 13 is reacted with a halide,such as elemental bromine, in an appropriate acid, such as hydrobromic,hydroiodic, or hydrochloric acid, in an appropriate acid based solvent,such as glacial acetic acid, to afford compound 14 (wherein Y is ahalide). Compound 14 is reacted with magnesium turnings to form aGrignard reagent, which is then reacted with an alkyl, heteroalkyl, analkenyl, a hetero-alkenyl, an alkynyl, or a heteroalkynyl containing analdehyde group, which can then subsequently converted into a goodleaving group and substituted with a hydride from a hydride donor, suchas sodium cyanoborohydride.

It should be understood many of the reagents and starting materials usedin the Schemes presented herein are readily available from variouscommercial suppliers, such as Sigma-Aldrich, Alfa Aesar, Tokyo ChemicalIndustry Co., LTD, etc. Moreover, many of these same reagents andstarting materials can be modified to incorporate additional functionalgroups by using standard organic synthesis reactions.

When a compound disclosed herein contains an acidic or basic moiety, itmay also disclosed as a pharmaceutically acceptable salt (See, Berge etal., J. Pharm. Sci. 1977, 66, 1-19; and “Handbook of PharmaceuticalSalts, Properties, and Use,” Stah and Wermuth, Ed.; Wiley-VCH and VHCA,Zurich, 2002).

Suitable acids for use in the preparation of pharmaceutically acceptablesalts include, but are not limited to, acetic acid, 2,2-dichloroaceticacid, acylated amino acids, adipic acid, alginic acid, ascorbic acid,L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoicacid, boric acid, (+)-camphoric acid, camphorsulfonic acid,(+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylicacid, cinnamic acid, citric acid, cyclamic acid, cyclohexanesulfamicacid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonicacid, 2-hydroxy-ethanesulfonic acid, formic acid, fumaric acid,galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid,D-glucuronic acid, L-glutamic acid, α-oxo-glutaric acid, glycolic acid,hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid,(+)-L-lactic acid, (+/−)-DL-lactic acid, lactobionic acid, lauric acid,maleic acid, (−)-L-malic acid, malonic acid, (+/−)-DL-mandelic acid,methanesulfonic acid, naphthalene-2-sulfonic acid,naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinicacid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid,pamoic acid, perchloric acid, phosphoric acid, L-pyroglutamic acid,saccharic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid,stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaricacid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, andvaleric acid.

Suitable bases for use in the preparation of pharmaceutically acceptablesalts, including, but not limited to, inorganic bases, such as magnesiumhydroxide, calcium hydroxide, potassium hydroxide, zinc hydroxide, orsodium hydroxide; and organic bases, such as primary, secondary,tertiary, and quaternary, aliphatic and aromatic amines, includingL-arginine, benethamine, benzathine, choline, deanol, diethanolamine,diethylamine, dimethylamine, dipropylamine, diisopropylamine,2-(diethylamino)-ethanol, ethanolamine, ethylamine, ethylenediamine,isopropylamine, N-methyl-glucamine, hydrabamine, 1H-imidazole, L-lysine,morpholine, 4-(2-hydroxyethyl)-morpholine, methylamine, piperidine,piperazine, propylamine, pyrrolidine, 1-(2-hydroxyethyl)-pyrrolidine,pyridine, quinuclidine, quinoline, isoquinoline, secondary amines,triethanolamine, trimethylamine, triethylamine, N-methyl-D-glucamine,2-amino-2-(hydroxymethyl)-1,3-propanediol, and tromethamine.

The disclosure provides that compounds disclosed herein can have prodrugforms. Prodrugs of the compounds are useful in the methods of thisdisclosure. Any compound that will be converted in vivo to provide abiologically, pharmaceutically or therapeutically active form of acompound of the disclosure is a prodrug. Various examples and forms ofprodrugs are well known in the art. Examples of prodrugs are found,inter alia, in Design of Prodrugs, edited by H. Bundgaard, (Elsevier,1985), Methods in Enzymology, Vol. 42, at pp. 309-396, edited by K.Widder, et al. (Academic Press, 1985); A Textbook of Drug Design andDevelopment, edited by Krosgaard-Larsen and H. Bundgaard, Chapter 5,“Design and Application of Prodrugs,” by H. Bundgaard, at pp. 113-191,1991); H. Bundgaard, Advanced Drug Delivery Reviews, Vol. 8, p. 1-38(1992); H. Bundgaard, et al., Journal of Pharmaceutical Sciences, Vol.77, p. 285 (1988); and Nogrady (1985) Medicinal Chemistry A BiochemicalApproach, Oxford University Press, New York, pages 388-392).

Prodrugs of compounds disclosed herein can be prepared by methods knownto one of skill in the art and routine modifications thereof, and/orprocedures found in U.S. Pat. No. 8,293,786, and references citedtherein and routine modifications made thereof.

In a certain embodiment, the disclosure provides for prodrug forms of acompound disclosed herein having a structure selected from the groupcomprising:

wherein, X is a pharmaceutically acceptable counter ion.

The disclosure also provides methods for identifying a library of Idmodulators comprising screening compounds of Formula I, II, II(a), III,or III(a) in assays. High throughput screening methodologies areparticularly envisioned for the detection of modulators of expression ofa target Id helix-loop-helix polypeptides, such as Id-1, using methodsalready described herein. Such high throughput screening methodstypically involve providing a combinatorial chemical or peptide librarycontaining a large number of potential therapeutic compounds (e.g.,modulator compounds). Such combinatorial chemical libraries or ligandlibraries are then screened in one or more assays to identify thoselibrary members (e.g., particular chemical species or subclasses) thatdisplay a desired characteristic activity. The compounds so identifiedcan serve as conventional lead compounds and/or used as potential oractual therapeutics. For administration to a subject, modulators of Idhelix-loop-helix expression and/or activity (e.g., inhibitory agents,nucleic acid molecules, proteins, or compounds identified as modulatorsof Id expression and/or activity) will preferably be incorporated intopharmaceutical compositions suitable for administration.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated either by chemical synthesis or biologicalsynthesis, by combining a number of chemical building blocks (e.g.,reagents such as amino acids). As an example, a linear combinatoriallibrary, e.g., a polypeptide or peptide library is formed by combining aset of chemical building blocks in every possible way for a givencompound length (e.g., the number of amino acids in a polypeptide orpeptide compound). Millions of chemical compounds can be synthesizedthrough such combinatorial mixing of chemical building blocks.

The preparation and screening of combinatorial chemical libraries iswell known to those having skill in the pertinent art. Combinatoriallibraries include, without limitation, peptide libraries (e.g., U.S.Pat. No. 5,010,175; Furka, 1991, Int. J. Pept. Prot. Res., 37:487-493;and Houghton et al., 1991, Nature, 354:84-88). Other chemistries forgenerating chemical diversity libraries can also be used. Nonlimitingexamples of chemical diversity library chemistries include peptoids (PCTPublication No. WO 91/019735), encoded peptides (PCT Publication No. WO93/20242), random bio-oligomers (PCT Publication No. WO 92/00091),benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993, Proc.Natl. Acad. Sci. USA, 90:6909-6913), vinylogous polypeptides (Hagiharaet al., 1992, J. Amer. Chem. Soc., 114:6568), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., 1992, J.Amer. Chem. Soc., 114:9217-9218), analogous organic synthesis of smallcompound libraries (Chen et al., 1994, J. Amer. Chem. Soc., 116:2661),oligocarbamates (Cho et al., 1993, Science, 261:1303), and/or peptidylphosphonates (Campbell et al., 1994, J. Org. Chem., 59:658), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (U.S. Pat. No. 5,539,083), antibody libraries(e.g., Vaughn et al., 1996, Nature Biotechnology, 14(3):309-314) andPCT/US96/10287), carbohydrate libraries (e.g., Liang et al., 1996,Science, 274-1520-1522) and U.S. Pat. No. 5,593,853), small organicmolecule libraries (e.g., benzodiazepines, Baum C&EN, Jan. 18, 1993,page 33; and U.S. Pat. No. 5,288,514; isoprenoids, U.S. Pat. No.5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; and the like).

In a certain embodiment, a compound disclosed herein can be administereddirectly or as a part of a composition. In other embodiments, thecomposition could be formulated as a pharmaceutical composition foradministration to a subject. In another embodiment, a compound disclosedherein can be a part of a pharmaceutical composition which includes oneor more pharmaceutically acceptable carriers. Pharmaceuticallyacceptable carriers include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

A pharmaceutical composition of the disclosure is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy to administer by a syringe. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it is preferable to include isotonic agents, for example, sugars,polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound, e.g. a compound disclosed herein, in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

In a particular embodiment, one or more compounds of the disclosure areprepared with carriers that will protect the compound against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations should be apparent to those skilled in the art. Thematerials can also be obtained commercially from Alza Corporation andNova Pharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the disclosure, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (e.g., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

Compositions and formulations of one or more compounds disclosed hereincan be used in combination with THC or a THC derivative to treat adisorder or disease in a subject. Examples of such disorders or diseaseswhich can be treated include cancer and other cell proliferativedisorders, such as chronic pancreatitis, psoriasis, neoplasms, angiomas,endometriosis, obesity, age-related macular degeneration, retinopathies,restenosis, scaring, fibrogenesis, fibrosis, cardiac remodeling,pulmonary fibrosis, scleroderma, and failures resulting from myocardialinfarction, keloids, fibroid tumors and stenting.

Moreover, a compound disclosed herein can have one or more biologicaleffects including, but not limited to, inducing apoptosis in malignantcells, inhibiting the proliferation of cancer cells, increasing theeffectiveness of chemotherapeutic agents, regulating transcriptionalactivity, reducing inflammation, increasing cellular differentiation,modulating ETS domain transcription factors, modulating PAXtranscription factors, modulating TCF-ETS domain transcription factors,down regulating RAF-1/MAPK, upregulating JNK signaling pathways, andmodulating cellular transformation. In a certain embodiment, thedisclosure provides for a composition comprising a compound disclosedherein that can be used to treat a disease or disorder which isameliorated by modulating ETS domain transcription factors, modulatingPAX transcription factors, modulating TCF-ETS domain transcriptionfactors, down regulating RAF-1/MAPK, and/or upregulating JNK signalingpathways.

In another embodiment, a method of treating cancer in a subjectcomprises administering to a subject in need of such treatment atherapeutically effective amount of a pharmaceutical compositionconsisting essentially of one or more compounds disclosed herein and apharmaceutically acceptable carrier.

In general, provided herein are methods for treating cancer byadministering to a subject a therapeutically effective amount of acomposition consisting essentially of a combination of one or morecompounds disclosed herein and THC or a derivative of THC. Examples ofTHC derivatives, includes, but are not limited to:Δ⁹-tetrahydrocannabinol-C₄, Δ⁹-tetrahydrocannabivarin,tetrahydrocannabiorcol, Δ⁹-tetrahydro-cannabinolic acid A,Δ⁹-tetrahydro-cannabinolic acid B, Δ⁹-tetrahydro-cannabinolic acid-C₄ A,Δ⁹-tetrahydro-cannabinolic acid-C₄ B, Δ⁹-tetrahydro-cannabivarinic acidA, Δ⁹-tetrahydro-cannabiorcolic acid A, Δ⁹-tetrahydro-cannabiorcolicacid B, (−)-Δ⁸-trans-(6aR,10aR)-Δ⁸-tetrahydrocannabinol,(−)-Δ⁸-trans-(6aR,10aR)-tetrahydrocannabinolic acid A, and(−)-(6aS,10aR)-Δ⁹-tetrahydrocannabinol. The methods include using apharmaceutical composition that includes a combination of one or morecompounds disclosed herein and THC or a THC derivative.

The compounds described herein and compositions comprising the compoundsare useful in modulating the expression and/or activity of Idpolypeptides in proliferating cells. In one exemplary embodiment, thedisclosure demonstrates a role for the compounds disclosed herein, ininhibiting the metastasis through inhibition of Id-1 expression and/oractivity. In a further embodiment, the disclosure demonstrates a rolefor the compounds disclosed herein, in inhibiting cell proliferation byactivating CB₂ receptors.

Accordingly, the compounds disclosed herein can be used in methods formodulating metastatic cancer cell progression by regulating theexpression and/or activity of an Id polypeptide. The methods includeusing a pharmaceutical composition that includes an agent that modulatesthe expression and/or activity of an Id polypeptide. Exemplary agentsinclude cannabinoids and derivatives arising there from, such ascannabidiol and derivatives therefrom.

U.S. patent application Ser. No. 11/390,682, and Internationalapplication No. PCT/US01/2881, are hereby incorporated by reference, intheir entirety for all purposes. While these publications providegeneral information about Id-1, it is understood that they do notpropose or describe the methods provided herein.

In a certain embodiment, the compounds disclosed herein can be usedalone or in combination with one or more additional therapeutic agents,such as THC. In a further embodiment, the compound disclosed herein canbe used in combination with THC or a derivative thereof in a definedratio based on weight. For example, in a certain embodiment, a compounddisclosed herein can be combined with THC or a derivative thereof, sothat the ratio of a compound disclosed herein to THC or a derivativethereof (wt/wt) is from 1:99, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40,70:30, 80:20, 90:10, 99:1, or any ratio in-between.

In a particular embodiment, the disclosure provides a composition fortreating cancer in a subject, the composition comprising a compounddisclosed herein and THC or a derivative thereof, such astetrahydrocannabivarin (THCV). In another embodiment, the disclosureprovides a method for treating a disease or disorder in a subject, suchas cancer, comprising administering to a subject in need of suchtreatment a therapeutically effective amount of a compound disclosedherein and THC or a THC derivative.

In another embodiment, a compound disclosed herein can be used to treata disease or disorder that would be benefited by modulating the activityof serotonin receptors, also known as 5-hydroxytryptamine receptors or5-HT receptors. In yet a further embodiment, a compound disclosed hereincan be used to treat a disease or disorder that would be benefited byactivating 5-HT receptors. In another embodiment, a compound disclosedherein can be used as an antidepressant, anxiolytic, or neuroprotectiveagent. In yet a further embodiment, a compound disclosed herein, can beused to relieve convulsion, inflammation, anxiety, and nausea.

In a certain embodiment, a compound disclosed herein can be used aloneor combined with THC or a derivative thereof to treat aneurodegenerative disease or disorder, including, but not limited to,Alzheimer's Disease, Parkinson's Disease, age related dementia,Huntington's Disease, and amyotrophic lateral sclerosis. In anotherembodiment, a compound disclosed herein can be used alone or combinedwith THC or a derivative thereof to treat pain or pain associated with adisease or disorder, including, but not limited to, pain associated withcancer, pain associated with arthritis, headaches, post-operative pain,fibromyalgia, and undiagnosed pain.

In another embodiment, a compound disclosed herein can be combined withone or more therapeutic agents that have one or more of the followingbiological effects, including, but not limited to, inducing apoptosis,regulating transcription, enhancing chemotherapy, reducing inflammation,promoting cellular differentiation, modulating cellular transformation,modulating cell migration, and/or inhibiting metastasis.

It should be understood that the administration of an additionaltherapeutic agent with a compound of the disclosure encompassesco-administration of these therapeutic agents in a substantiallysimultaneous manner, such as in a single capsule having a fixed ratio ofactive ingredients or in multiple, separate capsules for each activeingredient. In addition, administration of an additional therapeuticagent in combination with a compound disclosed herein also encompassesuse of each type of therapeutic agent in a sequential manner. In eithercase, the treatment regimen will provide beneficial effects of the drugcombination in treating the disorders described herein.

In a further embodiment, the compounds disclosed herein can be combinedwith one or more class of therapeutic agents, including, but not limitedto, alkylating agents, cancer immunotherapy monoclonal antibodies,anti-metabolites, mitotic inhibitors, anti-tumor antibiotics,topoisomerase inhibitors, photosensitizers, tyrosine kinase inhibitors,anti-cancer agents, chemotherapeutic agents, anti-migraine treatments,anti-tussives, mucolytics, decongestants, anti-allergic non-steroidals,expectorants, anti-histamine treatments, anti-retroviral agents, CYP3Ainhibitors, CYP3A inducers, protease inhibitors, adrenergic agonists,anti-cholinergics, mast cell stabilizers, xanthines, leukotrieneantagonists, glucocorticoid treatments, antibacterial agents, antifungalagents, sepsis treatments, steroidals, local or general anesthetics,NSAIDS, NRIs, DARIs, SNRIs, sedatives, NDRIs, SNDRIs, monoamine oxidaseinhibitors, hypothalamic phoshpholipids, anti-emetics, ECE inhibitors,opioids, thromboxane receptor antagonists, potassium channel openers,thrombin inhibitors, growth factor inhibitors, anti-platelet agents,P2Y(AC) antagonists, anti-coagulants, low molecular weight heparins,Factor VIa inhibitors, Factor Xa inhibitors, renin inhibitors, NEPinhibitors, vasopepsidase inhibitors, squalene synthetase inhibitors,anti-atherosclerotic agents, MTP inhibitors, calcium channel blockers,potassium channel activators, alpha-muscarinic agents, beta-muscarinicagents, anti-arrhythmic agents, diuretics, thrombolytic agents,anti-diabetic agents, mineralocorticoid receptor antagonists, growthhormone secretagogues, aP2 inhibitors, phophodiesterase inhibitors,anti-inflammatories, anti-proliferatives, antibiotics, farnesyl-proteintransferase inhibitors, hormonal agents, plant-derived products,epipodophyllotoxins, taxanes, prenyl-protein transferase inhibitors,anti-TNF antibodies and soluble TNF receptors, Cyclooxygenase-2inhibitors, and miscellaneous agents.

In yet a further embodiment, a compound disclosed herein can be combinedwith one or more classes of therapeutic agents, including, but notlimited to, alkylating agents, cancer immunotherapy monoclonalantibodies, anti-metabolites, mitotic inhibitors, anti-tumorantibiotics, topoisomerase inhibitors, photosensitizers, tyrosine kinaseinhibitors, anti-cancer agents, and chemotherapeutic agents.

In yet another embodiment, the additional therapeutic agent is ananti-cancer agent. Examples of anti-cancer agents include, but are notlimited to, methotrexate, fluorouracil, hydroxyurea, mercaptopurine,cisplatin, daunorubicin, doxorubicin, etoposide, vinblastine,vincristine, temozolomide, and pacitaxel.

In a particular embodiment, a pharmaceutical composition for treating acell proliferative disorder includes a compound disclosed herein and oneor more therapeutic agents selected from the group comprising: THC,paclitaxel, temozolomide, methotrexate, fluorouracil, hydroxyurea,mercaptopurine, cisplatin, daunorubicin, doxorubicin, etoposide,vinblastine, vincristine and pacitaxel.

For use in the therapeutic applications described herein, kits andarticles of manufacture are also described herein. Such kits cancomprise a carrier, package, or container that is compartmentalized toreceive one or more containers such as vials, tubes, and the like, eachof the container(s) comprising one of the separate elements to be usedin a method described herein. Suitable containers include, for example,bottles, vials, syringes, and test tubes. The containers can be formedfrom a variety of materials such as glass or plastic.

For example, the container(s) can comprise one or more compoundsdescribed herein, optionally in a composition or in combination withanother agent as disclosed herein. The container(s) optionally have asterile access port (for example the container can be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). Such kits optionally comprise a compound with anidentifying description or label or instructions relating to its use inthe methods described herein.

A kit will typically comprise one or more additional containers, eachwith one or more of various materials (such as reagents, optionally inconcentrated form, and/or devices) desirable from a commercial and userstandpoint for use of a compound described herein. Non-limiting examplesof such materials include, but are not limited to, buffers, diluents,filters, needles, syringes; carrier, package, container, vial and/ortube labels listing contents and/or instructions for use, and packageinserts with instructions for use. A set of instructions will alsotypically be included.

A label can be on or associated with the container. A label can be on acontainer when letters, numbers or other characters forming the labelare attached, molded or etched into the container itself, a label can beassociated with a container when it is present within a receptacle orcarrier that also holds the container, e.g., as a package insert. Alabel can be used to indicate that the contents are to be used for aspecific therapeutic application. The label can also indicate directionsfor use of the contents, such as in the methods described herein. Theseother therapeutic agents may be used, for example, in the amountsindicated in the Physicians' Desk Reference (PDR) or as otherwisedetermined by one of ordinary skill in the art.

EXAMPLES Cell Culture and Treatment of Breast Cancer Cell Lines

Human breast cancer cells lines MDA-MB231 and MDA-MB436 were obtainedfrom the ATCC. To prepare the MDA-MB231-Id-1 cells, cells were infectedwith a pLXSN-Id-1 sense expression vector. In all experiments, thedifferent cell populations were first cultured in RPMI media containing10% fetal bovine serum (“FBS”). On the first day of treatment the mediawas replaced with vehicle control or drug in RPMI and 0.1% FBS. Themedia with the appropriate compounds were replaced every 24 h. Δ⁹-THC,CBN, CBD, CBG, and CP55, 940 were obtained from NIH through the NationalInstitute of Drug Abuse. WIN 55, 212-2 was purchased from Sigma/RBI (St.Louis, Mo.).

Cell Culture and Treatment of GBM Cell Lines:

The human GBM cell lines used were SF126, U87 and U251. Cell lines weremaintained at 37° C. and 5% CO₂. In all experiments, the different cellpopulations were first cultured in RPMI media containing 10% FBS. On thefirst day of treatment the media was replaced with vehicle control ordrug in RPMI and 0.1% FBS. The media with the appropriate compounds werereplaced every 24 h. Δ⁹-THC, CBN, CBD, CBG, and CP55, 940 were obtainedfrom NIH through the National Institute of Drug Abuse. WIN55, 212-2 waspurchased from Sigma/RBI (St. Louis, Mo.).

MTT Assay:

To quantify cell proliferation, the3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrasodium bromide MTT assaywas used (Chemicon, Temecula, Calif.). Cells were seeded in 96 wellplates at 1×10³ cells/well for seven day experiments and 3×10³ cells percm² for three day experiments to obtain optimal cell density throughoutthe experiment. Upon completion of the drug treatments, cells wereincubated at 37° C. with MTT for four hours, and then isopropanol with0.04N HCl was added and the absorbance was read after one hour in aplate reader with a test wavelength of 570 nm. The absorbance of themedia alone at 570 nm was subtracted, and percent control was calculatedas the absorbance of the treated cells/control cells×100.

Statistical Analysis:

The IC₅₀ values with corresponding 95% confidence limits were comparedby analysis of logged data (GraphPad Prism, San Diego, Calif.). Whenjust the confidence limits of the IC₅₀ values overlapped significantdifferences were determined using unpaired Student's t-test. Wheresuitable, significant differences were also determined (Prism) usingANOVA or the unpaired Student's t-test. Bonferroni-Dunn post-hocanalyses were conducted when appropriate. p values <0.05 definedstatistical significance.

Apoptosis:

Cells were grown in 6 well culture dishes and were treated withcompounds of the disclosure every 24 hours for three days. The cellswere trypsinized, washed with PBS, and processed for labeling withfluorescein-tagged UTP nucleotide and PI by use of an Apo-Directapoptosis kit obtained from Phoenix Flow Systems (San Diego, Calif.) andwas used according to the manufacturer's protocol. The labeled cellswere analyzed by flow cytometry. Cell Flow Cytometry in combination withPI and annexin staining was used to quantify the percentage of cellsundergoing apoptosis in control and treatment groups. Percent controlwas calculated as annexin positive staining in treated cells/controlcells×100. PI staining was used to distinguish necrotic cells from thoseundergoing apoptosis.

Quantitative Western Analysis:

Cells were cultured and treated in 6-well dishes. After the cells werewashed twice with cold PBS, lysis buffer was added. The cells were thenlysed by freezing for 10 min at −70° C. and then thawing at ambienttemperature. The cell lysate was collected and the protein content wasdetermined by using Bradford reagent. Equal amounts of protein wereheated at 90° C. in Laemmli sample buffer which also included(3-mercaptoethanol. The samples were then loaded onto a precast SDS-PAGEgel (Bio-Rad Laboratories, Hercules, Calif.). After which, proteins werethen electroblotted onto an Immobilon membrane (Millipore, Billerica,Mass.) overnight at 2-4° C. The membrane was then blocked for 1 hourwith 5% nonfat dry milk which included TBS+Tween. The membranes werethen incubated with 1 mcg/mL of primary antibody (rabbitanti-phospho-JNK, rabbit anti-phospho-p38, rabbit anti-phospho-ERK1/2 orrabbit anti-ERK1/2; the antibodies were from Millipore) for 1 hour inblocking buffer. The blots were then washed three times with TBS+Tweenfor 10 min per wash. Secondary antibody (Donkey Anti-Rabbit IgG, fromJackson Immunoresearch, West Grove, Pa.) was then added. Alternatively,the primary antibody was anti-Id-1 and an appropriate secondary antibodywas used. Blots were incubated for 45 min and then washed 4 times withTBS+Tween for 15 min each. The blots were developed with SuperSignalFemto (Pierce, Rockford, Ill.), and imaged on either a Fluorchem 8900(Alpha Innotech, San Leandro, Calif.) or ECL Hyperfilm(Amersham-Pharmacia, Piscataway, N.J.). Band intensity values wereobtained (after background subtraction) directly from the Fluorchem 800using AlphaeaseFC software (San Leandro, Calif.) or from film usingImage-J (NIH, MD). As a normalization control for loading, blots werestripped and re-probed with mouse alpha-tubulin (Abcam, Cambridge,Mass.) and goat anti-mouse IgG (Jackson Immunoresearch) for the primaryand secondary antibodies, respectively.

Cell Cycle Analysis:

U251 cells were grown in Petri dishes (100 mm×15 mm) and received drugtreatments for 2 days. On the third day, the cells were harvested andcentrifuged at 1200 rpm for 5 minutes. The pellet was washed 1× withPBS+1% BSA, and centrifuged again. The pellet was re-suspended in 0.5 mlof 2% paraformaldehyde (diluted with PBS) and left to fix overnight atroom temperature. The next day the cells were pelleted and re-suspendedin 0.5 ml 0.3% Triton in PBS and incubated for 5 minutes at roomtemperature. The cells were then washed 2 times with PBS+1% BSA. Thecells were finally suspended in PBS (0.1% BSA) with 10 ug/ml PropidiumIodide and 100 μg/ml RNAse. The cells were incubated for 30 minutes atroom temperature before being stored at 4° C. Cell cycle was measuredusing a FACS Calibur using Cell Quest Pro and Modfit software.

Radical Oxygen Species (ROS) Measurements:

The production of cellular radical oxygen species (ROS)/H₂O₂ wasmeasured using 2′-7′Dichlorodihydrofluorescein (DCFH-DA, Sigma Aldrich).DCFH-DA is deacylated intracellularly into a non-fluorescent product,which reacts with intracellular ROS to produce 2′-7′dichlorofluorescein. 2′-7′Dichlorofluorescein remains trapped inside thecell, and can be measured quantitatively. U251 cells were plated onto 6well dishes and received drug treatments for three days. On the thirdday, 2 μM DCFH-DA was added to the media (MEM with 0.1% FBS) and thecells were incubated with DCFH-DA overnight. The next day, the cellswere trypsinized, washed with PBS, and the fluorescent intensity wasmeasured using a FACS and cell quest pro software.

Polymerase Chain Reaction:

Total cellular RNA was isolated from breast cancer cells treated withvehicle control or with CBD. Transcripts for Id-1 and for β-actin werereverse transcribed using SuperscriptII Reverse Transcriptasell(Gibco-BRL), and polymerase chain reaction performed. The 5′ and 3′ PCRprimers were AGGTGGTGCGCTGTCTGTCT (SEQ ID NO:1) and TAATTCCTCTTGCCCCCTGG(SEQ ID NO:2) for Id-1; and GCGGGAAATCGTGCGTGACATT (SEQ ID NO:3) andGATGGAGTTGAAGGTAGTTTCGTG (SEQ ID NO:4) for β-actin. PCR was performed inbuffer containing 1 μM of each of the 5′ and 3′ PCR primer and 0.5 U ofTaq polymerase using 25 cycles for amplification of Id-1 and β-actincDNAs. The cycle conditions were 45 sec denaturation at 94° C., 45 secannealing at 55° C., and 1 min extension at 72° C.

Id-1 Promoter Reporter Assays:

A SacI-BspHI fragment of 2.2 kb corresponding to the 5′ upstream regionof human Id-1 gene and driving a luciferase gene in a PGL-3 vector(Promega) was used (Id-1-sbsluc). Cells were plated in six well dishesin medium supplemented with 10% FBS and 5 μg/ml insulin. After 24 hours,cells were cotransfected with 6 μg of luciferase reporter plasmids and 2μg of pCMVβ (Clontech) using Superfect reagent (Qiagen). pCMVβ containedbacterial β-galactosidase and served to control for variation intransfection efficiency. 3 hours after transfection, the cells wererinsed twice with PBS and were cultured in the absence or presence ofCBD for 48-72 h. Cell pellets were lysed in 80 μl of reporter lysisbuffer (Promega) for 10 min at room temperature. Lysed cells werecentrifuged and supernatants harvested. Luciferase and β-gal assays wereperformed using Luciferase Assay System (Promega), β-Gal Assay Kit(Clontech) and a 2010 luminometer (Pharmingen).

In-Vivo Model of Breast Cancer Metastases:

4T1 cells were injected directly into the tail vein of syngeneic BALB/cmice. In this model, cancer cells have direct access to the blood streamresulting in a significant enhancement of lung metastasis and reducedvariability in the number of metastases formed as compared to orthotopicmodels. Two days after i.v. injection of 4T1 cells, the tumor bearingmice were injected i p. once a day with vehicle or compound (1 mg/kg).

Viability Assays Using Drug Combinations and Statistical Analysis:

Multiple viability assays in a 96 well format were run for each compoundand the average percent inhibition of cell viability was calculated andtransformed to fraction affected (Fa) i.e., percent inhibitory effect.Additional CI values and a dose reduction index (DRI) were alsocalculated using Compusyn software. After determining the(IC₅₀/Fa_(0.5)) values of the drugs individually, components were thencombined at the following concentration ranges: controls, 0.125×IC_(H),0.25×IC₅₀, 0.5×IC₅₀ and a combination index was calculated where CI<1,=1, and >1 indicate synergism, additive effect, and antagonism,respectively.

Antiproliferative MTT Studies using Breast Cancer MDA-MB231 andMDA-MB436 Cell Lines with WIN 55, 212-2 and CP55, 940, Δ⁹-THC, CBN, CBD,or CBG.

The antiproliferative activities of four groups of cannabinoid compoundswith MDA-MB231 and MDA-MB436 cells were examined. The groups included:(1) natural cannabis constituents that have affinity for C_(B1) andC_(B2) receptors, THC and CBN; (2) synthetic cannabinoid analogs thathave high affinity for C_(B1) and C_(B2) receptors, WIN 55, 212-2 andCP55, 940; and (3) natural cannabis constituents that do not haveappreciable affinity for C_(B1) and C_(B2) receptors, CBD and CBG.Breast cancer cells were treated for three days and IC₅₀ values werecalculated and provided in TABLE 1 below.

TABLE 1 Compound MDA-MB231 MDA-MB436 THC 1.2 (1.0-1.4) 2.5 (1.8-3.4) CBN1.2 (0.9-1.5) 2.6 (1.8-3.7) WIN 55, 212-2 1.7 (1.5-2.2) 2.4 (1.6-3.4) CP55, 940 2.5 (1.5-4.1) 1.3 (0.7-1.6) CBD 1.3 (1.0-1.9) 1.5 (1.0-1.9) CBG2.3 (2.1-2.5) 2.1 (1.5-3.0)

According to the IC₅₀ numbers (TABLE 1), the rank order of potencies forthe anti-proliferative effects of the cannabinoids in MDA-MB231 cellswas: CBD=THC=CBN>WIN55, 212-2>CBG=CP55, 940. The rank order of potenciesfor the antiproliferative effects of the cannabinoids in MDA-MB436 cellswas: CBD=CP55, 940>CBG=WIN55, 212-2=Δ⁹-THC=CBN. The data demonstratesthat cannabidiol (CBD) is an effective inhibitor of human breast cancercell aggressiveness, invasiveness, and therefore metastasis.

Boyden Chamber MDA-MB231 Cell Invasion Studies with CBD, Δ⁹-THC, or WIN55, 212-2.

Invasion is an important step towards breast cancer cell metastasis.Therefore, the effects of several cannabinoids were tested on theirability to modulate the migratory and invasiveness activity of the mostaggressive human breast cancer cell line, MDA-MB231, in a reconstitutedbasement membrane in a Boyden chamber. All three compounds tested, i.e.,CBD, THC, and WIN 55, 212-2, significantly reduced the invasion ofMDA-MB231 cells (FIG. 1, panel A). As was observed with the cellaggressiveness and invasiveness experiments, the most potent inhibitorout of the group of compounds, e.g., CBD, THC, and WIN 55, 212-2 forinvasion, was CBD. The IC₅₀ value and corresponding confidence limitsfor CBD were 260 nM (110-610).

Quantitative Western Analysis of Id-1 Expression in MDA-MB231 Cells withCBD, Δ⁹-THC, or WIN 55, 212-2.

The ability of CBD to regulate the expression of key genes that controlbreast cancer cell aggressiveness and invasiveness was determined. Apotential candidate protein that could mediate the effects of CBD onboth phenotypes was the helix-loop-helix protein Id-1. It was determinedthat treatment of MDA-MB231 cells with CBD led to aconcentration-dependent inhibition of Id-1 protein expression (FIG. 1,panel B and panel C). The inhibitory effect of CBD on Id-1 expressionoccurred at concentrations as low as 100 nM. CBD was more effective atreducing Id-1 protein expression compared to other cannabinoid compounds(FIG. 1, panel C). The CBD concentrations effective at inhibiting Id-1expression correlated with those used to inhibit the proliferative andinvasive phenotype of MDA-MB231 cells. Furthermore, the time periodneeded to observe the down-regulation of Id-1 protein in the presence ofCBD correlated with the inhibitory effects of CBD on the aggressivenessand invasiveness of MDA-MB231 cells (FIG. 1, panel D).

SAR Analysis of Id-1 Expression in MDA-MB231 Cells with (Abn)-CBDReceptor, Abn-CBD, 0-1602, CP55, 940, THC, CBN, or CBD.

In order to determine the structure activity relationship (“SAR”)between cannabinoids and the inhibition of Id-1, MDA-MB231 cells weretreated for two days with multiple cannabinoid compounds and Id-1protein levels were assessed. The compounds used included: (1) agonistsand antagonists to the putative abnormal (Abn)-CBD receptor, Abn-CBD and01602; (2) a synthetic cannabinoid analog that has high affinity for CB₁and CB₂ receptors, CP55, 940; (3) natural cannabis constituents thathave appreciable affinity for CB₁ and CB₂ receptors, Δ⁹-THC and CBN(FIG. 1, panel E). The greatest inhibition of Id-1 was observed in thepresence of CBD (FIG. 1, panel E). Also a small inhibition of Id-1 wasobserved in the presence of CP55, 940 (FIG. 1, panel E). No inhibitionof Id-1 was observed in the presence of THC, 0-1602 and Abn-CBD (FIG. 1,panel E).

The data demonstrates that CBD is an effective inhibitor of Id-1. Theinhibition of Id-1 does not appear to be related to the putative Abn-CBDreceptor. It also appears that the opened tetrahydropyran ring in CBD isonly partially responsible for its activity, since Abn-CBD and 0-1602did not inhibit Id-1. One potential key structure is the classicalcannabinoid aliphatic side chain: a region crucial for the activity ofnumerous classical and synthetic cannabinoids. CP55, 940 and CBN canpartially inhibit Id-1. In comparison to the classical cannabinoidstructure (Δ⁹-THC), each compound contains the side chain region, andthe cyclohexyl ring, however, the pyrane ring is removed in CP55, 940.CP55, 940 has an opened tetrahydropyran ring similar to CBD. The datasuggests that a general structural component of CBD, responsible forId-1 inhibition, is the combination of the opened tetrahydropyran ringand the classical cannabinoid aliphatic side chain.

Assays Using Id-1 Promoter Reporter Transfected MDA-MB231 Cells withCBD.

To determine if Id-1 represented a key mediator of CBD effects in highlyaggressive breast cancer cells, Id-1 was constitutively expressed intoMDA-MB231 cells (+)Id-1 as described in FIG. 2. The ectopic Id-1 gene,which is not under the control of the endogenous promoter, wasintroduced in the cells using the pLXSN retroviral vector. As a control,cells were infected with an empty pLXSN vector (−)Id-1. In controlcells, treatment with CBD led to a significant reduction in cellinvasiveness (FIG. 2, panel A (upper panels) and FIG. 2, panel B).Western blotting confirmed the down-regulation of Id-1 expression inthis control cell line (FIG. 2, panel C). In contrast to these results,CBD did not inhibit cell invasiveness (FIG. 2, panel A (lower panels)and FIG. 2, panel B) or Id-1 expression (FIG. 2, panel C) inMDA-MB231(+)Id-1 cells that ectopically expressed Id-1.

Gene Expression Assays Looking at Id-1 Gene Expression with CBD inMDA-MB231 Cells.

Referring to FIG. 3, panel A, Id-1 mRNA expression was significantlyreduced upon treatment with CBD. To determine if this effect was due tothe inhibition of transcription, a construct was used that contained theId-1 promoter fused to a luciferase reporter in a PGL-3 basic vector.This construct was transiently transfected into MDA-MB231 cells.Twenty-four hours after transfection, MDA-MB231-Id-1-luc cells weretreated with CBD for 2 or 3 days and luciferase activity was measured(FIG. 3, panel B and panel C). Transfection efficiency and analysis ofequal amounts of total protein were controlled by cotransfection of thecells with pCMVB containing β-galactosidase. Treatment with CBD resultedin a significant inhibition of luciferase activity. This effect wastime-dependent with the greatest inhibition occurring on day 3. Thesefindings correlated to the data obtained when the expression of the Id-1protein was assessed by Western analysis.

CBD Increases Survival in a Syngeneic Mouse Model of Breast cancer.

While CBD has been shown to inhibit breast cancer metastasis in vivo,pharmacological analysis to determine potency and efficacy has notperformed. The 4T1 intravenous (i.v.) mouse model of breast cancermetastasis to assess the activity of CBD in vivo was utilized. Incomparison to orthotopic models, when 4T1 cells are injected i.v.through the tail vein, there is a rapid disease progression (2 weeks)and high penetrance of 4T1 tumor formation to the lung which greatlylimits variability. This model is therefore highly suited for theanalysis of drug potency and efficacy, including the assessment ofsurvival. CBD produced a robust dose-dependent inhibition of metastaticspread of 4T1 cells to the lung in vivo (FIG. 4, panel A). One day afteri.v. injection of the breast cancer cells, mice were treated daily fortwo weeks by intra-peritoneal injection with vehicle (used as a control)or CBD at a dose range of 0.1 to 5 mg/kg. CBD inhibited total breastcancer metastasis up to 75% with an EC_(H) value of 0.3 mg/kg(CI=0.2-0.5). In both the intravenous and orthotopic models, CBD washighly effective at targeting metastatic foci >2 mm (FIG. 4, panel B).

Based on the robust inhibition of lung tumor formation produced by CBD,it was predicted that treatment with the drug would increase survival intumor bearing mice. A survival study was carried out (FIG. 4, panel C).One day after i.v. injection of 4T1 cells, mice were treated daily withvehicle or 1 mg/kg CBD (a dose producing maximum anti-metastaticactivity) until they demonstrated signs of disease progression thatnecessitated euthanasia (as described in the Methods section). In thishighly aggressive mouse model of breast cancer, CBD produced a mediansignificant increase in survival of seven days (p<0.006).

CBD Down-Regulates Id-1 Expression and Breast Cancer Cell Proliferationin Lung Metastatic Foci.

Id-1 was a key factor whose expression needed to be down-regulated inorder to observe the effects of CBD on the reduction of breast cancercell aggressiveness in vitro. Lung tissue from mice treated with vehicleor CBD (1 mg/kg) were evaluated for Id-1 expression and Ki67, a markerof cellular proliferation (FIG. 5, panel A). Treatment with CBD produceda significant down-regulation of Id-1 expression; 60.9% of lung foci invehicle-treated mice were strongly positive, while only 5.6% of the lungfoci were labeled in the CBD-treated group (FIG. 5, panel B). CBD alsoproduced a significant down-regulation of Ki67 demonstrating its abilityto reduce tumor cell proliferation in metastatic foci (FIG. 5, panel C).

CBD was Found to Inhibit Id-1 and Ki67 Expression in Metastic Foci fromLung Tissue Isolated from Mice.

Immunohistochemical detection of Id-1 and Ki67 was performed in lungtissues of vehicle and CBD treated mice. Nuclei were stained withhematoxylin. As seen in the panels, upper panels are ×200 magnificationand lower panels are ×400, CBD treated cells had noticeably lowerhematoxylin staining levels than Vehicle treated cells (FIG. 6, panelA). The intensity of the immunohistochemical (IHC) detection of Id-1 wasthen graded from 0 to 4. It was found that CBD treated cells hadsignificantly higher amounts of grade 0, grade 1, grade 2 cells, whilevehicle had higher amounts of grade 3 and grade 4 cells (FIG. 6, Panel B(left). When the data was presented as a statistical analysis, CBDtreated cells averaged around 1 and vehicle treated cells averagedaround 2.8 (FIG. 6, Panel B (right)). The percentage of Ki67 positivecells per lung metastatic foci was then evaluated. It was determinedthat there was a significantly higher percent of ki67 positive cells inVehicle treated versus CBD treated (FIG. 6, Panel C).

CBD Produces a Dose-Dependent Inhibition of Metastasis in More AdvancedStages of Breast Cancer Progression.

While CBD was effective at reducing the total number of metastatic focithat formed in the lung, it was significantly more active at targetingmetastatic foci ≧2 mm. This suggested the compound could be effective atinhibiting the growth of secondary tumors even after their initialestablishment in lung. To determine whether CBD could inhibit theformation of lung tumor foci in more advanced stages of metastasis, micewere treated at a time point where visual lung metastatic foci werealready formed (as shown at day seven in FIG. 7, panel A). Mice wereinjected i.v. with 4T1 cells were kept for one week in order to allowfor the formation of visible lung metastatic foci. The mice were thentreated with CBD. CBD dose-dependently reduced the growth of establishedlung metastatic foci and reduced the formation of new metastatic foci(FIG. 7, panel B and panel C). Using a dose of CBD that produced maximuminhibition of metastasis in this model (FIG. 7, panel B), the ability ofthe cannabinoid to increase survival was also assessed (FIG. 7, panelD). Treatment of mice with CBD (1 mg/kg) starting on day seven alsoincreased survival. While the median increase in survival was only aday, a subset of animals did live three to five days longer (p<0.02).

In-Vivo Studies of Breast and Brain Tumor Formation with CBD.

The compounds of the disclosure were effective in inhibiting tumorformation in an in-vivo model. Breast cancer 4T1 cells weresubcutaneously injected into the mammary fat pad of BALBc mice (FIG. 8,Panel A), or into the flank of Nude mice (FIG. 8, Panel B). After oneweek post injection, the mice were treated daily (systemic in A, andperitumoral in B) with CBD (mg/kg). Primary tumor volume was thencalculated by measuring the perpendicular largest diameters of the tumorwith a caliper and using the formula (L×W²/2). The tumor proliferationin-vivo data demonstrates that CBD is a potent inhibitor of tumorproliferation (FIG. 8, panel A and B), and in some cases completelyeradicated tumors (FIG. 8, panel C).

In-Vivo Metastatic Studies Using a Xenograph Mouse Model for HumanBreast Cancer with CBD.

It was found that mice treated with CBD showed a significant reductionin the number of lung metastatic foci in comparison to mice treated withvehicle alone. Lung metastases were generated in Nude mice after tailvein injection of 1×10⁶ MDA-MB231-luc-D3H2LN cells. One day after theinjection, the tumor bearing mice were injected i.p. once a day withvehicle or 0.5 mg/kg and 1 mg/kg CBD for three weeks. Visible lungmetastases were then counted and measured by using a dissectingmicroscope. By comparing the percent of metastasis in mice treated withCBD versus mice trated with vehicle alone (100% metastatsis), micetreated with CBD demonstrated at least a 70% reduction in the number ofmetastases (FIG. 9).

In-Vivo Late Stage Metastatic Studies Using a Xenograph Mouse Model forHuman Breast Cancer with CBD.

CBD was found to inhibit the formation of metastatic foci in laterstages of metastatic progression in mice which were injected with 4T1cells. Lung metastases were generated in BALB/c mice after tail veininjection of 20×10⁵ mouse 4T1 cells. As 1 mm³ tumors were first detectedusing a dissecting scope on day 7 (FIG. 10, panel A), this day waschosen to initiate treatments. One week after the tail vein injection of20×10⁵ mouse 4T1 cells, the tumor bearing mice were injected i.p. once aday with vehicle or 1 mg/kg and 10 mg/kg CBD for seven days. Visiblelung metastases were counted and measured by using a dissectingmicroscope. By comparing the percent of metastasis in mice treated withCBD versus mice trated with vehicle alone (100% metastatsis), micetreated with CBD demonstrated at least a 70% reduction in the number ofmetastases (FIG. 10, panel B).

Cell Viability Studies with 4T1 Breast Cancer Cells Using CBD inCombination with Paclitaxel.

It was found that CBD can enhance the ability of Paclitaxel to inhibitthe viability of 4T1 breast cancer cells. The concentration responsecurves were first generated for Paclitaxel (PAC), CBD alone, and acombination of CBD and Paclitaxel (FIG. 11, panel A). The inhibitoryvalues from the concentration response curves were then used tocalculate combination index (CI) values at multiple combination ratios(FIG. 11, panel B). From which, the data was also used to calculate i)IC50 values, the slope of the curve (m) and a goodness of fit value (r).It was found that CBD acts synergistically with Paclitaxel (FIG. 11,panel C).

Quantitative Western Analysis of Id-1 Expression in U251 Cells with orwithout CBD Treatment.

To examine whether CBD would downregulate Id-1 protein expression in aGBM based cell line, quantitative Western analysis was performed withU251 cells. It was determined that treatment of U251 cells with CBD ledto a concentration-dependent inhibition of Id-1 protein expression inU251 GBM cells (FIG. 12).

Quantitative Western Analysis of Id-1 Expression in Different Types ofCancer Cells with or without CBD Treatment.

Quantitative Western analysis was performed to examine whether CBD woulddownregulate Id-1 protein expression in cell lines for breast, prostate,salivary gland, head and neck, and glioblastoma cell lines. It wasdetermined that treatment of cells with CBD (1.5 μM) led to significantinhibition of Id-1 protein expression in the breast cancer cell lines,MDA-MB231 and MDA-MB436 (FIG. 13, panel A); prostate cancer cell linesPC3 and DU145 (FIG. 13, panel B); head and neck cancer cell line, SAS(FIG. 13, panel C (right)); and glioblastoma cell lines, U251 and SF126(FIG. 13, panel D). For the salivary gland cancer cell line, ACCM, ahigher concentration of CBD (2.5 μM) was required to see a significantdownregulation in Id-1 expression (FIG. 13, panel C (left)).

Antiproliferative MTT Studies using GBM SF126, U87 and U251 Cell Lineswith WIN 55, 212-2 or Δ⁹-THC.

Two commonly used CB₁ and CB₂ receptors agonists were chosen to studythe effect of cannabinoid treatment on the growth of three humanglioblastoma multiforme (GBM) cell lines. THC, a natural cannabisconstituent, and WIN 55, 212-2, a synthetic cannabinoid analog, havehigh affinity for CB₁ and CB₂ receptors. Human GBM cells were treatedwith multiple concentrations of THC and WIN 55, 212-2. Cellproliferation was measured using the MTT assay and corresponding IC₅₀values were calculated in three GBM cell lines over a seven daytreatment (TABLE 2). SF126 cells overall were most sensitive to theantiproliferative effects of THC and WIN 55, 212-2. Cannabidiol (CBD), anatural cannabis compound that does not have appreciable affinity forCB₁ and CB₂ receptors, was also tested in the GBM cell line, SF126. TheIC₅₀ value was 0.73 μM (0.64-0.82). Data are the means and corresponding95% confidence limits of at least three experiments. IC₅₀ values arereported in pM.

TABLE 2 Compound SF126 U87 U251 THC  0.9 (0.7-1.4)  1.6 (1.0-2.4) 1.1(0.84-1.4) WIN 55, 0.84 (0.74-0.95) 0.77 (0.65-0.90) 1.1 (0.97-1.3)212-2 CBD 0.73 (0.64-0.82)

Antiproliferative MTT Studies using GBM SF126 Cell Line WIN 55, 212-2and CP55, 940, Δ⁹-THC, CBN, CBD, or CBG.

Treatment periods were shortened to three days during experiments withadditional agonists since significant effect were observed at this timepoint. Three groups of cannabinoid compounds were chosen for a broaderanalysis of antiproliferative activity in the single GBM cell line,SF126. (1) Natural cannabis constituents that have affinity for CB₁ andCB₂ receptors, THC and CBN. (2) Synthetic cannabinoid analogs that havehigh affinity for CB₁ and CB₂ receptors, WIN 55, 212-2 and CP55, 940.(3) Natural cannabis constituents that do not have appreciable affinityfor CB₁ and CB₂ receptors, CBD and CBG. IC₅₀ values for theantiproliferative effects of cannabinoid agonists on SF126 cell growthover a three day treatment were obtained (TABLE 3). SF126 cells weretreated with a range of concentrations of multiple cannabinoid agonists,and the corresponding IC₅₀ values were calculated. Cell proliferationwas assessed using the MTT assay. Data are the means and corresponding95% confidence limits of at least three experiments. IC₅₀ values arereported in pM.

TABLE 3 Compound SF126 THC 2.5 (1.8-3.4) CBN 1.2 (0.9-1.6) WIN 55, 212-21.3 (1.2-1.4) CP 55, 940 3.3 (2.9-3.7) CBD 1.2 (1.1-1.3) CBG 1.6(1.5-1.7)

The rank order of potencies was: CBD=CBN=WIN 55, 212-2>CBG>THC=CP55940.Again, CBD was one of the most potent compounds tested.

Antiproliferative MTT Studies using GBM SF126, U87 and U251 Cell Lineswith CBD.

Invasion is also an important step towards brain cancer progression. Thedisclosure also provides methods and compositions for the treatment ofbrain cancer progression. Therefore, the ability of CBD to reduce thegrowth and invasiveness activity of glioblastoma muliforme (GBM) cancercells was tested. Multiple glioblastoma muliforme (GBM) cell lines weretreated for three days. IC₅₀ values for the antiproliferative effects ofCBD were calculated in multiple GBM cell lines over a three daytreatment. Cell proliferation was assessed using the MTT assay. Data arethe means and corresponding 95% confidence limits of at least threeexperiments. IC₅₀ values are reported in μM in TABLE 4 below.

TABLE 4 Cell Line CBD IC₅₀ SF126 1.2 (1.1-1.3) U87 0.7 (0.5-1.0) U2510.6 (0.5-0.7)It was determined that U251 cells were the most sensitive to theantiproliferative activity of CBD. CBD was also able to significantlyreduce the invasiveness of U251 cells.

Effects of CBD on GBM Cell Invasion in In Vivo Studies.

To determine whether CBD could inhibit GBM cell invasion through intactbrain tissue, an organotypic brain slice assay was utilized. GFP-labeledU251 cells were treated for two days in culture with vehicle or CBD. Onthe third day, the cells were harvested and transferred to the top of a0.5 mm coronal rat brain slice obtain from a postnatal day 3 rat pup. Aporous cell culture insert containing the slice was suspended for threedays in a well containing conditioned media with either vehicle or 1 μMCBD. After three days, GFP-labeled cells that successfully invadedthrough the slice are visualized using an inverted microscope and wefound that CBD was highly effective at inhibiting invasion of U251 cellsthrough the organotypic brain slice (FIG. 14, panel A). GBM tumors weregenerated in nu/nu mice by intracranial injection of U251 GBM cells. 7days after tumor implantation, mice were injected systemically(intraperitoneal) with 15 mg/kg CBD 5 days a week for 28 days untilvehicle-treated animals demonstrated signs of significant diseaseprogression (hunched posture and reduced mobility), when all mice in thestudy were euthanized in order to compare tumor growth. CBD produced arobust reduction of GBM progression, decreasing the tumor area by 95%(FIG. 14, panels B and C). In one of the five mice treated with CBD, notumor cells were observed in any of the brain regions analyzed. Targetvalidation showed that, in tumors responding to treatment, CBD produceda significant down-regulation of Id-1 expression (FIG. 14, panel D).This occurred concomitantly with a decrease in tumor cell proliferation(the number of Ki67-positive nuclei was decreased by 87%±9 (p<0.0001,Student's t-test)) (FIG. 14, panel D).

Measuring the Effect of CBD on the Aggressiveness of Head and Neck asWell as Salivary Gland Cancer Cells.

Head and neck cancer cell lines (SAS and HSC-2 in the upper panels) andsalivary gland cancer cell lines (ACCM and ACC2 in the lower panels)were treated with CBD. Id-1 gene expression was downregulated in allcell lines (FIG. 15, panel A). CBD had a significant effect on SAS tumorcell viability and invasiveness (FIG. 15, panel B (upper panels)). CBDalso had a significant effect on ACCM tumor cell viability andinvasiveness (FIG. 15, panel B (lower panels)). There was a significantreduction in the number and size of lung metastases after mice wereinjected with ACCM cells and treated daily with CBD (FIG. 15, panel C).The number of lung metastatic foci after injection of ACCM cells wassignificantly reduced upon CBD treatment (FIG. 15, panel D). The numberof metastatic foci greater than >1 mm was also significantly reducedupon treatment with CBD (FIG. 15, panel D, (right)).

Cell Viability Studies with U251 GBM Cells Using CBD in Combination withTemozolomide.

CBD was found to enhance the ability of Temozolomide to inhibit theviability of U251 GBM cells. The concentration response curves werefirst generated for temozolomide, CBD alone, and a combination of CBDand temozolomide (FIG. 16, panel A). The inhibitory values from theconcentration response curves were then used to calculate combinationindex (CI) values at multiple combination ratios (FIG. 16, panel B).From which, the data was also used to calculate (i) IC₅₀ values, theslope of the curve (m) and a goodness of fit value (r). It was foundthat CBD acts synergistically with Temozolomide (FIG. 16, panel C).

Boyden Chamber U251 Cell Invasion Studies with CBD and/or Δ⁹-THC.

In addition to uncontrolled cell growth, a hallmark phenotype ofaggressive GBM tumor cells is their ability to migrate away for theprimary tumor of origin and invade into neighboring CNS tissue.Experiments were performed to determine whether the addition of CBD toΔ⁹-THC would improve the activity of the compound to inhibit migrationand invasion through a reconstituted basement membrane in a Boydenchamber. Δ⁹-THC effectively inhibited the invasiveness of U251 cells(FIG. 17). Additionally, Δ⁹-THC was significantly more potent atinhibiting U251 cell invasiveness in comparison to the inhibition ofcell growth and induction of apoptosis. The predicted IC₅₀ for theability of Δ⁹-THC to inhibit U251 cell invasiveness was 85 nM (49-150).Whereas both THC and CBD were able to inhibit U251 cell invasiveness,the combined addition of the compounds did not result in activitysuggesting a synergistic interaction (FIG. 17).

2×2 Antiproliferative MTT Assays using Gliobastoma U251, U87, and SF126Cell Lines with Δ⁹-THC and/or CBD.

Non-psychoactive cannabinoids, compounds that do not interactefficiently with CB₁ and CB₂ receptors, can modulate the actions of THC.The experiments described below, using multiple human glioblastomamultiforme (“GBM”) cells lines, compared the antiproliferative activityof non-psychoactive cannabinoids to synthetic and natural CB₁ and CB₂agonists. The activity of THC was tested in combination with CBD. InU251 and SF126 cell lines, THC in combination with a lower concentrationof CBD, acted synergistically to inhibit GBM cell growth and induceapoptosis. The inhibitory properties of the combination were the resultof activation of CB₂ receptors and a corresponding increase in oxygenradical formation. The signal transduction mechanisms associated withthe effects of the combination treatment were different from thoseobserved with the individual compounds. The disclosure demonstrates thatthe addition of CBD to THC improves the overall potency and efficacy ofΔ⁹-THC in the treatment of patients with cell proliferative disorderssuch as, for example, GBM.

Treatment groups were divided into (1) no treatment (control), (2) THCalone, (3) CBD alone, (4) THC and CBD combined. Positive and negativeaspects of constituent interaction were determined in this 2×2 designusing 2-way analysis of variance as described by. In the proliferationassays, IC₅₀ values with corresponding 95% confidence limits werecalculated using non-linear analysis of logged data (GraphPad Prism, SanDiego, Calif.). Significant differences were also determined using ANOVAwhere suitable. Bonferroni-Dunn post-hoc analyses were conducted whenappropriate. p values <0.05 defined statistical significance.

Three GBM cell lines, SF126, U251 and U87 cells, were used to determinethe effects of combination treatments. When applied in combination, THCand CBD produces synergistic inhibition of cell growth in SF126 and U251cells but not in U87 cells (FIG. 18, panels A, B, and C). Concentrationsof THC and CBD alone that produce only minimal effects on cellproliferation were combined and further tested in a 2×2 factorial designin the positive responding cell lines (SF126 and U251) (FIG. 18, panelsD, E, and F). The most pronounced synergistic activity was observed withU251 cells, therefore, this cell lines was used to determine themechanism of action for the combination effect. The 4:1 (1.7 μM: 0.4 μM)ratio of THC and CBD was used for the following experiments.

It was determined that THC and CBD act synergistically to inhibit thegrowth of multiple GBM cell lines. It has been suggested thatnon-psychoactive cannabinoid constituents can either potentiate orinhibit the actions of THC. The CB₁ and CB₂ receptor agonist, THC, caninhibit GBM growth in vitro and in vivo. CBD, a cannabinoid constituentwith negligible affinity for CB₁ and CB₂ receptors can also inhibit thegrowth of GBM in vitro and in vivo. The disclosure demonstrates that ofthe non-psychoactive cannabinoids, CBD is a far superior inhibitor ofGBM cell growth.

Quantitative Western Analysis of pERK, JNK, and p38 MAPK Expression inU251 Cells with CBD and/or Δ⁹-THC.

The disclosure demonstrates that the combination treatment of Δ⁹-THC andCBD leads to the modulation of specific mitogen activated kinases(MAPK). The regulation of ERK, JNK, and p38 MAPK activity plays acritical role in controlling cell growth and apoptosis. Therefore inU251 cells, it was determined whether treatment with a combination ofTHC and CBD could alter the activity of ERK, JNK, and p38 MAPK.Treatment with the combination of cannabinoids led to a profounddown-regulation of p-ERK but no significant change in total ERK (FIG.19, panel A). Additionally, no inhibition of JNK or p38 MAPK activitywas observed (FIG. 19, panel B). When U251 cells were treated withindividual concentration of THC and CBD, instead of the combination, nochanges in pERK were observed (FIG. 19, panel C).

Apoptosis Studies Using U251 Cells with Δ⁹-THC and/or CBD.

The disclosure further demonstrates that the combination treatment ofTHC and CBD induces apoptosis. Significant reductions in ERK activityhave been shown to lead to induction of apoptosis. The large reductionin GBM cell growth and ERK activity, observed in the presence of thecombination treatment of THC and CBD, suggested there would be acorresponding modulation of the cell cycle and programmed cell death.Therefore, U251 cells were treated with THC and CBD alone or with thecombination of the two, and cell cycle was analyzed using cell flowcytometery (FIG. 20). The combination of THC and CBD produced anincrease in the population of cells in G1 phase and a decrease in cellsin S phase. Additionally, there was an increase in the population ofcells in the G2/M phase. These changes in G1, S and G2/M phase arehallmarks of cell cycle arrest.

When administered separately, THC (1.7 μM) and CBD (0.4 μM) bothproduced increases in the population of cells in G1 and G2/M phase anddecreases in cells in S phase. Albeit, the magnitude of these effectswas reduced compared to those observed with the combination treatment.By measuring annexin concentration, a large increase in apoptosis wasobserved when THC and CBD were combined (FIG. 21). Separately THC (1.7μM) and CBD (0.4 μM) did not produce significant changes in apoptosis(FIG. 21).

The Combination Treatment of Δ⁹-THC and CBD Produced the Activation ofMultiple Caspases.

Caspases play a primary role in the regulation of programmed cell death.Therefore, multiple caspase pathways were evaluated to determinemechanisms by which the combination treatment increased apoptosis.Treatment with the combination of THC and CBD led to a significantup-regulation of caspase 3, 7, and 9 activities as well as an increasein PARP (FIG. 22). Small increases in the activity of caspase 7, caspase9 and PARP but not caspase 3 were observed when U251 cells were treatedwith the individual concentration of THC (FIG. 22). In the presence ofCBD alone no changes in caspase activity were observed (FIG. 22).

Apoptosis Induced by the Combination of THC and CBD can be at LeastPartially Blocked by Administering Cannabinoid Receptor Antagonists.

Apoptosis produced by the combination of THC and CBD was partiallyblocked by the CB₂ receptor antagonist, SR144528, but complete reversalwas observed in the presence of the anti-oxidant, α-tocopherol (TCP)(FIG. 23, panel A). The concentrations of the individual cannabinoids(THC and CBD) were next increased in order to attempt to match levels ofapoptosis produced by the combination treatment. The purpose of theseexperiments was to determine whether the compounds alone recruitedsimilar pathways as compared to the combination of THC and CBD. WhenU251 cells were treated with THC alone, the induction of apoptosis wascompletely blocked by α-tocopherol and partially blocked by the CB2antagonist, SR144528 (FIG. 23, panel B). However, THC alone could notproduce the level of apoptosis observed with the combination treatment(FIG. 23, panel A and B). This finding was not simply an issue of thetreatment concentration used since continuing to increase levels of THCdid not produce a greater induction of apoptosis. When U251 cells weretreated with CBD alone, the induction of apoptosis was completelyblocked by α-tocopherol but no reversal was observed with SR144528 (FIG.23, panel C); this result would be expected since CBD does not interactefficiently with either CB₁ or CB₂ receptors.

The ability of the higher concentrations of THC and CBD alone to inhibitp-ERK were also studied and compared to the combination treatment (FIG.23, panel D). Again, the combination treatment produced a profound downregulation of p-ERK. However, the higher concentration of Δ⁹-THC alonehad no effect on p-ERK activity. The higher concentration of CBDproduced a small inhibition of p-ERK. This suggests the pathway(s)activated by the THC and CBD combination that leads to p-ERKdown-regulation, is unique to the combination treatment. As predicted byα-tocopherol blockade, the combination of THC and CBD produced asignificant increase in the formation of ROS as assessed by DCDHF-DAoxidation (FIG. 23, panel E).

A wide range of cannabinoids inhibit the proliferation of human GBMcells. In addition to testing THC, the analysis included thenon-psychoactive cannabis constituents CBD, CBN and CBG. Overall, CBDwas the most potent inhibitor tested.

Combining THC and CBD together resulted in a synergistic increase in theinhibition GBM growth and produced significant increases in apoptosis.This synergistic activity occurred in two of three GBM cell linestested. The synergistic inhibition of GBM cell growth was in part theresult of a greater amount of apoptosis being produced in presence ofthe combination compared to administration of THC alone. Treatment ofU251 cells with the combination of cannabinoids led to a profounddown-regulation of ERK activity, but not p38 MAPK and JNK1/2. Thereduction of ERK activity was specific for the combination treatmentindicating that all the effects observed were not simply due to anincrease in potency of THC upon co-application with CBD. The specificreduction in ERK activity, observed in the presence of the combinationtreatment, may be one of the primary mechanisms leading to thesynergistic increase in inhibition of GBM cell growth and the inductionof apoptosis. THC was also effective at inhibiting the invasiveness ofU251 cells. However, there was no suggestion of a synergisticinteraction upon addition of CBD.

ROS Measurement Studies Using GBM Cells with THC and/or CBD.

The disclosure demonstrates that the synergistic inhibitory effects ofcombination treatment are the result of CB₂ receptor activation andproduction of oxygen radicals. Depending on the cancer cell line andcompound used, studies have linked the inhibitory activity ofcannabinoids to activation of CB₁, CB₂, vanilloid (VR1) receptors, andthe production of oxygen radicals. An increase in apoptosis produced bythe combination of THC/CBD was partially dependent on CB₂ receptoractivation. Apoptosis produce by treatment of Δ⁹-THC alone was alsopartially dependent on CB₂ receptor activation. Importantly, theinduction of apoptosis in the presence of the combination treatment wassignificantly greater than that observed with THC alone. Apoptosisproduced by CBD in U251 cells was not dependent CB₂ receptor activation.Comparable results with CBD were also observed using another GBM cellline, SF126. Apoptosis produced by the combination of THC and CBD wasgreatly dependent on the production of oxidative stress and resulted inthe activation of both extrinsic and extrinsic caspase pathways.

Δ⁹-THC and CBD activate unique pathways in GBM cells that ultimatelyculminate in inhibition of cancer cell growth and invasion as well asinduction of cell death. The data presented here show that thesynergistic activity of the combination treatment is due in part to aspecific convergence of distinct pathways controlled by the individualcompounds. This convergence of inhibitory pathways unique to THC and CBDleads to an overall synergistic reduction of GBM cell growth andsurvival. Combinations, compared to individual drug treatments withspecific cannabinoid-based compounds may represent a significantimprovement for the treatment of patients with GBM. These synergisticeffects may also be present in additional cancers. With the discovery ofa specific molecular mechanism potentially explaining the synergisticeffects, additional combination treatments may able to be refined inorder to further improve antitumor activity.

Development Novel CBD Derivatives with Enhanced Anti-NeoplasticActivities.

The previous data suggest key structural requirements of CBD impart theability to inhibit Id-1 expression. It was also shown that classical CB₁and CB₂ receptor agonists (e.g. THC) do not target Id-1. CB₂ selectivecannabinoid agonists, having limited activity at CB₁ receptors(psychoactivity), but sharing structural similarities with CBD weredeveloped to modulate Id-1 expression more effectively than CBD.Moreover, these novel CBD based analogs would not only effectivelytarget Id-1 but would also have the advantage of targeting two distinctcannabinoid antitumor pathways leading to enhanced antitumor activity.Two separate screens of selective resorcinol derivatives were performedby testing for selectivity to CB₂ receptors and for their ability toinhibit MDA-MB231 cell viability (TABLE 5, summary). In the firstscreen, a candidate compound O-1422 was found to be as potent as CBD. Inthe second screen, a candidate compound O-1663 was found to be 2.2 foldmore active than CBD (TABLE 5). The structure of O-1442 and O-1663 arealmost identical with the exception of one R group. A smaller subset ofthe same analogs, including O-1442 and O-1663, were further screened fortheir ability to inhibit 4T1 breast cancer cell viability inanticipation of running in vivo experiments in immune competent mice(TABLE 5). Similar activities were observed in both MDA-MB231 and 4T1cells. While both compounds were selective for targeting CB₂ receptorsover CB₁ receptors, O-1442 still had a relatively high affinity for CB₁receptors (potential for significant psychoactivity) and was less potentthan the O-1663 CBD analog at inhibiting breast cancer cell viability.The O-1663 CBD analog was found to have a lower affinity for CB₁receptors in comparison to THC and produced little activity in thetetrad assay-measure of psychoactive properties of cannabinoids. Basedupon the data presented in TABLE 5, the O-1663 CBD analog was evaluatedin additional studies.

TABLE 5 Cell line Compound IC₅₀ Confidence Limits MDA-MB231 THC 3.0 μM(2.3-3.9) MDA-MB231 CBD 1.9 μM (1.5-2.5) MDA-MB231 O-1663 ^(#)0.85 μM(0.79-0.91) MDA-MB231 O-1422 1.7 μM (1.5-2.0) MDA-MB231 O-2137 45%^(a)MDA-MB231 O-1657 39%^(a) MDA-MB231 O-1424 22%^(a) MDA-MB231 O-3853 3%^(a) MDA-MB231 O-2981 15%^(a) MDA-MB231 O-2988 26%^(a) MDA-MB231O-5788  0%^(a) MDA-MB231 O-5832  0%^(a) MDA-MB231 O-4233  0%^(a)MDA-MB231 O-5881  0%^(a) 4T1 THC 2.3 μM (1.9-2.7) 4T1 CBD 1.8 μM(1.2-2.7) 4T1 O-1663 ^(#)0.83 μM (0.79-0.87) 4T1 O-1422 4.5 μM (1.5-12) 4T1 O-2137 3.5 μM (1.8-7.0) 4T1 O-3853  7%^(a) 4T1 O-5788  0%^(a) Inrespect to TABLE 5, data represent the mean with correspondingconfidence limits for 3-5 independent confidence limits for 3-5independent determinations. ^(a)Percent inhibition of cell viabilityproduced at 5 μM. ^(#)Statistically significant increase in potencycompared to CBD (p < 0.05)

Antiproliferative MTT Studies Using Breast Cancer MDA-MB231 Cell Linewith CBD and O-1663 CBD Analog.

In further cell viability in-vitro studies with CBD, a novel syntheticCBD derivative, O-1663 CBD analog, was found to be unexpectedly farsuperior to CBD in inhibiting human breast cancer cell and glioblastomaaggressiveness, invasiveness, and therefore metastasis (FIG. 24, panelsA and B). The IC₅₀ values were calculated and provided in TABLE 6 below.

TABLE 6 Compound MDA-MB231 CBD 2.2 (1.9-2.6) O-1663 CBD analog 1.2(0.9-2.0)

Antiproliferative MTT Assays Using the GBM U251 Cell Line with CBD orCBD 1663 Analog.

As with the MDA-MB231 cell line, CBD 1663 analog exhibits a strongerinhibitory antiproliferative effect than CBD in the GBM U251 cell line,by a surprisingly large margin (FIG. 24, Panel B). The IC₅₀ values werecalculated and provided in TABLE 7 below.

TABLE 7 Compound U251 CBD  2.9 (2.7-3.2) O-1663 CBD analog 0.53(0.54-0.56)

Quantitative Western Analysis of Id-1 Expression in MDA-MB231 Cells andU251 Cells with CBD or O-1663 CBD Analog.

To examine whether O-1663 CBD analog's potent inhibitory effect oncancer cell proliferation was associated with downregulating Id-1protein expression, quantitative Western analysis for Id-1 proteinexpression was performed. MDA-MB231 breast cancer cells and U251 GBMcells were treated with 1.0 μM of cannabinoid for two days and thenanalyzed for Id-1 protein expression by quantitative Western analysis.It was determined that cancer cells treated with the 0-1663 CBD analogdownregulated Id-1 protein expression to a far greater extent than CBD(FIG. 24, panel C).

Boyden Chamber MDA-MB231 Cell Invasion Studies with CBD and O-1663 CBDAnalog.

Boyden chamber studies for invasion by MDA-MB231 or U251 cells wereperformed with either CBD or O-1663 CBD analog. The O-1633 CBD analogwas found to be far superior to CBD in inhibiting invasion by thecancerous cells (FIG. 24, panel D).

O-1663 CBD Analog but not CBD Reduces Breast Cancer Cell AggressivenessThrough the Activation of CB₂ Receptors.

To Directly test whether the O-1663 CBD analog could co-target twodistinct cannabinoid antitumor pathways, the effects of CBD and theO-1663 CBD analog on cell viability with multiple antagonists wereprobed (FIG. 25). The ROS scavenger, a-TOC was able to reverse theinhibitory effects of CBD on cell viability. A CB₁ receptor antagonisthad no effect on reversing the activity of CBD (FIG. 25, panels A andB). While a subtle reversal of CBD activity was observed with the CB₂receptor antagonists SR14458 it was not significant (FIG. 25, panels Aand B). In contrast, the CB₂ receptor agonist SR14458 was able topartially reverse the inhibitory effects of the O-1663 CBD analog onbreast cancer cell viability (FIG. 25, panels A and B). The ability ofthe O-1663 CBD analog and CBD to inhibit invasion was next compared(FIG. 25, panel C). O-1663 was 1.7 fold more potent than CBD atinhibiting the invasion of MDA-MB231 cells. The IC₅₀ value andcorresponding confidence limits for the O-1663 CBD analog and CBD were0.6 μM (0.5-0.7) and 1 μM (0.8-1.2), respectively.

O-1663 CBD Analog is More Potent than CBD at Inhibiting Id-1 Expressionand Up-Regulating ROS.

Whereas a CBD (1 μM) produced a partial reduction of Id-1 expression,treatment with the same concentration of the O-1663 CBD analog producedalmost a complete down-regulation of Id-1 expression in both MDA-MB231and 4T1 cells (FIG. 25, panel 4). Id-2 is a marker of good prognosis inbreast cancer patients, is important for the maintenance of adifferentiated and noninvasive phenotype in breast cancer cells, and isup-regulated following inhibition of Id-1. As demonstrated in FIG. 25,panel D, the O-1663 CBD analog was more potent than CBD at up-regulatingId-2 expression in breast cancer cells demonstrating specificity fortargeting Id-1. In culture, CBD-induced generation of ROS is a primarymechanism that leads to inhibition of cell growth, invasion, andsurvival across multiple cancers. The generation of ROS by CBD leads tothe inhibition of Id-1 expression in breast cancer cells. Using theapproximate IC₅₀ for CBD and THC for inhibition of cell proliferationand viability, it was found that CBD produced a robust up-regulation ofROS whereas THC produced no significant increase in ROS (FIG. 26, panelA). The ability of CBD and the O-1663 CBD analog to stimulate theproduction of ROS was next compared. It was found that the O-1663 CBDanalog was significantly more potent and efficacious than CBD atgenerating ROS. The involvement of CB₂ receptors in the productions ofROS produced by CBD and the O-1663 CBD analog was next investigated. Inthe presence of SR14458, there was a small but significant reversal ofROS whereas >50% of the ROS produced by the O-1663 CBD analog wasreversed in the presence of SR14458 (FIG. 26, panel B).

O-1663 CBD Analog but not CBD Up-Regulates p8 and Autophagy.

A primary mechanism for the antitumor activity of the mixed CB₁ and CB₂receptor agonist (THC) and CB₂ selective agonists is the up-regulationof p8 and the autophagy pathway. The O-1663 CBD analog was able toup-regulate p8 and the marker of autophagy LC3 (FIG. 26, panels C andD). The O-1663 CBD analog was also significantly more potent that THC atup-regulating p8 and L3. In comparison to THC and the O-1663 CBD analogthe IC₅₀ of CBD for inhibiting cell viability, that is sufficient tostimulate ROS and down-regulate Id-1 expression, did not produceup-regulation of p8 and LC3.

In Comparison to CBD, the O-1663 CBD Analog is More Active at TargetingBreast Cancer Metastasis.

Since O-1663 CBD analog was significantly more effective than CBD attargeting Id-1 and also targets CB₂ receptor antitumor activity, it waspredicted that the compound would be more active in inhibitingmetastasis of breast cancer cell lines which are dependent Id-1expression for disease progression (e.g., 4T1 and MDA-MB231). Theactivity of CBD to the 0-1663 CBD analog in the 4T1 i.v. model of breastcancer metastasis was then compared (FIG. 27, panels A and B). We foundthat the 0-1663 CBD analog was a 2.3 fold more potent at inhibitingtotal metastasis [O-1663: EC₅₀=0.13 (0.10-0.20); CBD: EC₅₀=0.29(0.18-0.49)] and 7.0 fold more potent than CBD at inhibiting lungmetastatic foci >2 mm [O-1663: EC₅₀=0.02 (0.01-0.05); CBD: EC₅₀=0.15(0.10-0.24)]. At the most effective dose of O-1663 CBD analog (1 mg/kg)only one of five animals treated with the O-1663 CBD analog had a singlemetastatic foci greater that >2 mm. The 0-1663 CBD analog was also morepotent than CBD in the human MDA-MB231 breast cancer model of metastasis(FIG. 27, panel C). No overt toxicity was noted with the O-1663 CBDanalog in the mouse models of metastasis as assessed by weight,appearance and generally activity.

The Anti-Metastatic Activity of the O-1663 CBD Analog but Not CBD isPartially Reversed by a CB₂ Receptor Antagonist.

In culture and in vivo, the data supports that the O-1663 CBD analogtargets two distinct antitumor tumor pathways. Those targeted by CB₂receptor activation such as p8 and autophagy, and those specific to CBDsuch as up-regulation of ROS and down-regulation of Id-1. To providefurther support for this hypothesis in vivo, mice bearing 4T1 tumorswere treated with CBD and the O-1663 CBD analog in the presence of a CB₂receptor antagonist (FIG. 27, panels D and E). In agreement with the invitro results, the antimetastic activity of CBD was not affected byco-administration with SR14458 whereas the anti-metastatic activity ofthe O-1663 CBD analog was partially reversed by the antagonist. Inaddition, a combination treatment with CBD and THC was included. Thecombination of CBD with THC produced the same level of anti-metastaticactivity as the O-1663 CBD analog.

O-1663 CBD Analog Produces a Robust Inhibition of More Advanced Stagesof Metastasis and Increases Survival.

The activity of CBD and the O-1663 CBD analog to inhibit the formationof lung tumor foci in more advanced stages of metastasis was thencompared. Mice were treated at a time point where visual lung metastaticfoci were already formed. Mice were injected i.v. with 4T1 cells andkept for one week in order to allow for the formation of visible lungmetastatic foci. The mice were then treated with CBD and the O-1663 CBDanalog. As demonstrated in FIG. 28, panels A and B, both cannabinoidsdose-dependently reduced the growth of established lung metastatic fociand reduced the formation of new metastatic foci. The O-1663 CBD analogwas significantly more potent than CBD in reducing percent metastasis.Since the O-1663 CBD analog produced a robust inhibition of totalmetastasis and was highly active at reducing metastatic foci >2 mm, itwas predicted that the O-1663 CBD analog would produce substantialincreases in survival even when administered at more advanced stages ofbreast cancer metastasis. A survival study was then carried out wherethe activity of CBD to O-1663 CBD analog was compared (FIG. 28, panelC). Seven days after i.v. injection of 4T1 cells, mice were treateddaily with vehicle, CBD (0.5 or 1 mg/kg, a dose producing maximumanti-metastatic activity) or O-1663 CBD analog (0.5 or 1 mg/kg). Themice were treated until they demonstrated signs of disease progressionthat necessitated euthanasia, as previously described. CBD produced amedium increase in survival of 4 days (p<0.02) whereas the O-1663 CBDanalog produced a medium increase in survival of 30 days (p<0.006). Inthe group treated with the O-1663 CBD analog, 50% of the mice were stillalive and demonstrated no signs of disease progression at time ofeuthanasia (2 months). More importantly, under a dissection microscope,visible lung metastatic foci were not present in 20% of the mice.

Quantitative Western Analysis of Id-1 Expression in Breast Cancer 4T1Cells with CBD or O-1663 CBD Analog.

To examine whether CBD or O-1663 CBD analog would inhibit Id-1 proteinexpression in cells proposed for the in-vivo metastic cancer model,quantitative Western analysis was performed with 4T1 cells. 4T1 cellswere treated with 1.0 μM of cannabinoid for two days and then analyzedfor Id-1 protein expression. It was determined that 4T1 cells treatedwith the O-1663 CBD analog downregulated Id-1 protein expression to afar greater extent than CBD (FIG. 29, panel A).

In-Vivo Studies of Breast Cancer Metastases with CBD or O-1663 CBDAnalog.

4T1 cells were injected into the tail vein of syngeneic BALB/c mice andthen were intraperitoneal injected 2 days later with either vehicle, CBD(1 mg/kg), or O-1663 CBD analog (1 mg/kg). Treatment with CBD and CBD1663 analog resulted in a reduction of the total amount of metastaticfoci. As with the in-vitro data, the in-vivo data demonstrates that theO-1663 CBD analog is a more potent inhibitor than CBD for cancerproliferation (FIG. 29, panels B and C).

In-Vivo Advanced Stage Metastasis Survivability Studies in a Mouse Modelwith CBD or O-1663 CBD Analog.

To determine whether cannabinoids could inhibit the formation of lungtumor foci in more advanced stages of metastasis, we treated mice at atime point where visual lung metastatic foci were already formed (FIG.30, panel A). Mice injected tail vein with 4T1 cell were kept for oneweek in order to allow for the formation of visual lung metastatic focimeasuring up to 1 mm. The mice where then treated with drug for twomonths. During this time period, mice were removed from the study whenthey demonstrated signs of disease progression that necessitatedeuthanasia. As demonstrated in FIG. 30, panel B, the O-1663 CBD analogwas significantly more active than CBD at increasing overall survival.In this highly aggressive syngeneic mouse model of breast cancer, theO-1663 CBD analog produced a significant increase in survival (p<0.006,Log-rank (Mantel-Cox) Test). Whereas the average median survival for thecontrol group and CBD was 32 and 38 days, respectively, 50% of theanimals treated with the O-1663 CBD analog were still alive at thecompletion of the study.

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the apparatus, systems and methods of thedisclosure, and are not intended to limit the scope of what theinventors regard as their disclosure. Modifications of theabove-described modes for carrying out the disclosure that are obviousto persons of skill in the art are intended to be within the scope ofthe following claims.

A number of embodiments of the disclosure have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the disclosure.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A compound having the structure of Formula II:

or a pharmaceutically acceptable salt, or prodrug thereof, wherein: X isindependently C or N; R²-R³ are each independently selected from thegroup consisting of hydroxyl, (C₁-C₂)alkoxy, carboxylic acid, amine,halo, cyano, and (C₁-C₃) ester; R⁴-R⁵ are each independently selectedfrom the group consisting of hydrogen, deuterium, hydroxyl,(C₁-C₂)alkoxy, carboxylic acid, amine, halo, cyano, and (C₁-C₃)ester; R⁶is selected from the group consisting of an unsubstituted (C₁-C₁₂)alkyl, an unsubstituted hetero (C₁-C₁₁) alkyl, an unsubstituted (C₁-C₁₂)alkenyl, an unsubstituted hetero (C₁-C₁₁) alkenyl, an unsubstituted(C₁-C₁₂)alkynyl, and an unsubstituted hetero(C₁-C₁₁)alkynyl; R¹⁰-R¹⁹ areeach independently selected from the group consisting of hydrogen,deuterium, functional group (“FG”), optionally substituted (C₁-C₈)alkyl,optionally substituted hetero(C₁-C₈)alkyl, optionally substituted(C₁-C₈)alkenyl, optionally substituted hetero(C₁-C₈)alkenyl, optionallysubstituted (C₁-C₈)alkynyl, optionally substituted hetero(C₁-C₈)alkynyl,optionally substituted (C₅-C₁₂)cycloalkyl, optionally substituted(C₅-C₁₂)cycloalkenyl, optionally substituted (C₅-C₁₂)cycloalkynyl,optionally substituted (C₄-C₁₁)heterocycle, optionally substituted aryl,and optionally substituted extended mixed ring system; and R²⁰ isselected from the group consisting of optionally substituted(C₅-C₁₂)cycloalkyl, optionally substituted (C₅-C₁₂)cycloalkenyl,optionally substituted (C₅-C₁₂)cycloalkynyl, optionally substituted(C₄-C₁₁)heterocycle, substituted aryl, optionally substituted arylhaving two or more rings, and optionally substituted extended mixed ringsystem.
 2. The compound of claim 1, wherein R⁶ is selected from thegroup consisting of methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl,n-heptyl, isopropyl, sec-butyl, (1-methyl)butyl, (1-methyl)pentyl,(1-methyl)hexyl, (1-methyl)heptyl, (1,1-dimethyl)propyl,(1,1-dimethyl)butyl, (1,1-dimethyl)pentyl, (1,1-dimethyl)hexyl,(1,1-dimethyl)heptyl, (1,2-dimethyl)propyl, (1,2-dimethyl)butyl,(1,2-dimethyl)pentyl, (1,2-dimethyl)hexyl, (1,2-dimethyl)heptyl,(1,3-dimethyl)butyl, (1,3-dimethyl)pentyl, (1,3-dimethyl)hexyl,(1,3-dimethyl)heptyl, (1,4-dimethyl)pentyl, (1,4-dimethyl)hexyl,(1,4-dimethyl)heptyl, (1,5-dimethyl)hexyl, (1,5-dimethyl)heptyl,(1,6-dimethyl)heptyl, (1,2-diethyl)butyl, (1,2-diethyl)pentyl,(1,2-diethyl)hexyl, (1,2-diethyl)heptyl, (1,2-diethyl)pentyl,(1,3-diethyl)pentyl, (1,3-diethyl)hexyl, (1,3-diethyl)heptyl,(1,4-diethyl)pentyl, (1,4-diethyl)hexyl, (1,4-diethyl)heptyl,(1,5-diethyl)hexyl, (1,5-diethyl)heptyl, (1,6-diethyl)heptyl,(1,2,3-trimethyl)butyl, (1,1,2-trimethyl)butyl, (1,1,3-trimethyl)butyl,(1,2,3-trimethyl)pentyl, (1,1,2-trimethyl)pentyl,(1,1,3-trimethyl)pentyl, (1,2,4-trimethyl)pentyl,(1,3,4-trimethyl)pentyl, (1,1,4-trimethyl)pentyl,(1,2,3-trimethyl)hexyl, (1,1,2-trimethyl)hexyl, (1,1,3-trimethyl)hexyl,(1,2,4-trimethyl)hexyl, (1,2,5-trimethyl)hexyl, (1,1,4-trimethyl)hexyl,(2,3,4-trimethyl)hexyl, (2,3,5-trimethyl)hexyl, (1,1,5-trimethyl)hexyl,(1,2,3-trimethyl)heptyl, (1,1,2-trimethyl)heptyl,(1,1,3-trimethyl)heptyl, (1,2,4-trimethyl)heptyl,(1,1,5-trimethyl)heptyl, (1,1,6-trimethyl)heptyl,(1,2,5-trimethyl)heptyl, (1,2,6-trimethyl)heptyl,(2,3,4-trimethyl)heptyl, (2,3,5-trimethyl)heptyl,(2,3,6-trimethyl)heptyl, (2,4,5-trimethyl)heptyl,(2,4,6-trimethyl)heptyl, (3,4,5-trimethyl)heptyl,(3,4,6-trimethyl)heptyl, and (4,5,6-trimethyl)heptyl.
 3. The compound ofclaim 1, wherein R²⁰ is selected from the group consisting of anoptionally substituted (C₅-C₇)cycloalkyl, an optionally substituted(C₅-C₇)cycloalkenyl, a substituted aryl, an optionally substituted arylhaving two or more rings, and an optionally substituted heterocyclecontaining 4, 5, or 6 ring atoms.
 4. The compound of claim 3, whereinthe optionally substituted heterocycle is selected from the groupconsisting of:


5. The compound of claim 3, wherein the substituted aryl and optionallysubstituted aryl having two or more rings is selected from the groupconsisting of:


6. The compound of claim 3, wherein the optionally substituted(C₅-C₇)cycloalkyl is selected from the group consisting of:


7. A compound having the structure of Formula III:

or a pharmaceutically acceptable salt, or prodrug thereof, wherein: X isindependently either a C or N; R²-R³ are each independently a hydroxylor (C₁-C₂)alkoxy; R⁶ is selected from the group consisting of anunsubstituted (C₁-C₁₂) alkyl, an unsubstituted hetero (C₁-C₁₁) alkyl, anunsubstituted (C₁-C₁₂) alkenyl, an unsubstituted hetero (C₁-C₁₁)alkenyl, an unsubstituted (C₁-C₁₂)alkynyl, and an unsubstitutedhetero(C₁-C₁₁)alkynyl; R¹¹-R¹⁸ are each independently selected from thegroup consisting of hydrogen, deuterium, FG, optionally substituted(C₁-C₈)alkyl, optionally substituted hetero(C₁-C₈)alkyl, optionallysubstituted (C₁-C₈)alkenyl, optionally substituted hetero(C₁-C₈)alkenyl,optionally substituted (C₁-C₈)alkynyl, optionally substituted hetero(C₁-C₈) alkynyl; and R²¹-R³¹ are each independently selected from thegroup consisting of hydrogen, deuterium, FG, optionally substituted(C₁-C₈)alkyl, hetero (C₁-C₈) alkyl, optionally substituted(C₁-C₈)alkenyl, optionally substituted hetero(C₁-C₈)alkenyl, optionallysubstituted (C₁-C₈) alkynyl, optionally substituted hetero (C₁-C₈)alkynyl, optionally substituted (C₅-C₈)cycloalkyl, optionallysubstituted (C₅-C₈)cycloalkenyl, optionally substituted(C₅-C₈)cycloalkynyl, optionally substituted (C₄-C₈)heterocycle,optionally substituted aryl, and optionally substituted extended mixedring system.
 8. A compound of claim 7, wherein the compound is

or a pharmaceutically acceptable salt, or prodrug thereof.
 9. A compoundof claim 8, wherein the prodrug form of the compound is selected fromthe group consisting of:

wherein, X is a pharmaceutically acceptable counter ion.
 10. A compoundselected from the group consisting of:


11. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier together with a compound of claim
 1. 12. Thepharmaceutical composition of claim 11, wherein the composition furthercomprises an additional therapeutic agent.
 13. The pharmaceuticalcomposition of claim 12, wherein the additional therapeutic agent isΔ⁹-tetrahydrocannabinol (“THC”) or a THC derivative.
 14. Thepharmaceutical composition of claim 13, wherein the THC derivative isselected from the group consisting of Δ⁹-tetrahydrocannabinol-C₄,Δ⁹-tetrahydrocannabivarin, tetrahydrocannabiorcol,Δ⁹-tetrahydro-cannabinolic acid A, Δ⁹-tetrahydro-cannabinolic acid B,Δ⁹-tetrahydro-cannabinolic acid-C₄ A, Δ⁹-tetrahydro-cannabinolic acid-C₄B, Δ⁹-tetrahydro-cannabivarinic acid A, Δ⁹-tetrahydro-cannabiorcolicacid A, Δ⁹-tetrahydro-cannabiorcolic acid B,(−)-Δ⁸-trans-(6aR,10aR)-Δ⁸-tetrahydrocannabinol,(−)-Δ⁸-trans-(6aR,10aR)-tetrahydrocannabinolic acid A, and(−)-(6aS,10aR)-Δ⁹-tetrahydrocannabinol.
 15. The pharmaceuticalcomposition of claim 12, wherein the additional therapeutic agent isselected from the group consisting of alkylating agents, cancerimmunotherapy monoclonal antibodies, anti-metabolites, mitoticinhibitors, anti-tumor antibiotics, topoisomerase inhibitors,photosensitizers, tyrosine kinase inhibitors, anti-cancer agents,chemotherapeutic agents, anti-migraine treatments, anti-tussives,mucolytics, decongestants, anti-allergic non-steroidals, expectorants,anti-histamine treatments, anti-retroviral agents, CYP3A inhibitors,CYP3A inducers, protease inhibitors, adrenergic agonists,anti-cholinergics, mast cell stabilizers, xanthines, leukotrieneantagonists, glucocorticoid treatments, antibacterial agents, antifungalagents, sepsis treatments, steroidals, local or general anesthetics,NSAIDS, NRIs, DARIs, SNRIs, sedatives, NDRIs, SNDRIs, monoamine oxidaseinhibitors, hypothalamic phoshpholipids, anti-emetics, ECE inhibitors,opioids, thromboxane receptor antagonists, potassium channel openers,thrombin inhibitors, growth factor inhibitors, anti-platelet agents,P2Y(AC) antagonists, anti-coagulants, low molecular weight heparins,Factor VIa inhibitors, Factor Xa inhibitors, renin inhibitors, NEPinhibitors, vasopepsidase inhibitors, squalene synthetase inhibitors,anti-atherosclerotic agents, MTP inhibitors, calcium channel blockers,potassium channel activators, alpha-muscarinic agents, beta-muscarinicagents, anti-arrhythmic agents, diuretics, thrombolytic agents,anti-diabetic agents, mineralocorticoid receptor antagonists, growthhormone secretagogues, aP2 inhibitors, phophodiesterase inhibitors,anti-inflammatories, anti-proliferatives, antibiotics, farnesyl-proteintransferase inhibitors, hormonal agents, plant-derived products,epipodophyllotoxins, taxanes, prenyl-protein transferase inhibitors,anti-TNF antibodies and soluble TNF receptors, and Cyclooxygenase-2inhibitors.
 16. The pharmaceutical composition of claim 15, wherein theadditional therapeutic agent is selected from the group consisting ofalkylating agents, cancer immunotherapy monoclonal antibodies,anti-metabolites, mitotic inhibitors, anti-tumor antibiotics,topisomerase inhibitors, photosensitizers, tyrosine kinase inhibitors,anti-cancer agents, and chemotherapeutic agents.
 17. The pharmaceuticalcomposition of claim 16, wherein the additional therapeutic agent is ananti-cancer agent.
 18. The pharmaceutical composition of claim 17,wherein the anti-cancer agent is paclitaxel and/or temozolomide.
 19. Amethod for modulating helix-loop-helix Id protein expression, cellproliferation, cell invasion, metastasis or a combination thereof invivo and/or in vitro by administering a compound of claim
 1. 20. Amethod for treating a disease or disorder in a subject, comprisingadministering to a subject a therapeutically effective amount of acompound of claim 1, wherein the disease or disorder can be amelioratedby inhibiting the expression of an Id polypeptide, by activatingcannabinoid type 2 (“CB₂”) receptors or a combination thereof.
 21. Themethod of claim 20, wherein the disease or disorder is selected from thegroup consisting of cancer, chronic pancreatitis, psoriasis, neoplasms,angiomas, endometriosis, obesity, age-related macular degeneration,retinopathies, restenosis, scaring, fibrogenesis, fibrosis, cardiacremodeling, pulmonary fibrosis, scleroderma, failure associated withmyocardial infarction, keloids, fibroid tumors, stenting, Alzheimer'sDisease, Parkinson's Disease, age related dementia, Huntington'sDisease, and amyotrophic lateral sclerosis.
 22. The method of claim 21,wherein cancer is selected from the group consisting of leukemia,melanoma, squamous cell carcinoma (SCC), hepatocellular carcinoma,colorectal adenocarcinoma, pancreatic cancer, lung cancer, kidneycancer, medullary thyroid cancer, papillary thyroid cancer, astrocytictumor, neuroblastoma, Ewing's sarcoma, ovarian tumor, cervical cancer,endometrial carcinoma, breast cancer, prostate cancer, and malignantseminoma.
 23. The method of claim 22, wherein the cancer is breastcancer.
 24. The method of claim 22, wherein the cancer is brain cancer.25. The method of claim 24, wherein the brain cancer is glioblastomamultiforme.
 26. A method of treating a disease or disorder in a subject,comprising administering to a subject a therapeutically effective amountof a compound having the structure of:

or a pharmaceutically acceptable salt, or prodrug thereof, wherein thedisease or disorder can be ameliorated by inhibiting the expression ofan Id polypeptide, by activating CB₂ receptors or a combination thereof.27. The method of claim 26, wherein the disease or disorder is selectedfrom the group consisting of leukemia, melanoma, SCC, hepatocellularcarcinoma, colorectal adenocarcinoma, pancreatic cancer, lung cancer,kidney cancer, medullary thyroid cancer, papillary thyroid cancer,astrocytic tumor, neuroblastoma, Ewing's sarcoma, ovarian tumor,cervical cancer, endometrial carcinoma, breast cancer, prostate cancer,and malignant seminoma.
 28. The method of claim 27, wherein the diseaseor disorder is breast cancer or brain cancer.
 29. The method of claim28, wherein the brain cancer is glioblastoma multiforme.
 30. The methodof claim 26, wherein the method further comprises administering to thesubject one or more alkylating agents, cancer immunotherapy monoclonalantibodies, anti-metabolites, mitotic inhibitors, anti-tumorantibiotics, topoisomerase inhibitors, photosensitizers, tyrosine kinaseinhibitors, anti-cancer agents, chemotherapeutic agents, or acombination thereof.